Arginine methyltransferase inhibitors and uses thereof

ABSTRACT

Described herein are compounds of Formula (I), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds of the present invention are useful for inhibiting arginine methyltransferase activity. Methods of using the compounds for treating arginine methyltransferase-mediated disorders are also described.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional patent application, U.S. Ser. No. 61/781,063, filed Mar. 14,2013, the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Epigenetic regulation of gene expression is an important biologicaldeterminant of protein production and cellular differentiation and playsa significant pathogenic role in a number of human diseases.

Epigenetic regulation involves heritable modification of geneticmaterial without changing its nucleotide sequence. Typically, epigeneticregulation is mediated by selective and reversible modification (e.g.,methylation) of DNA and proteins (e.g., histones) that control theconformational transition between transcriptionally active and inactivestates of chromatin. These covalent modifications can be controlled byenzymes such as methyltransferases (e.g., arginine methyltransferases),many of which are associated with specific genetic alterations that cancause human disease.

Disease-associated chromatin-modifying enzymes (e.g., argininemethyltransferases) play a role in diseases such as proliferativedisorders, autoimmune disorders, muscular disorders, vascular disorders,metabolic disorders, and neurological disorders. Thus, there is a needfor the development of small molecules that are capable of inhibitingthe activity of arginine methyltransferases.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Arginine methyltransferases are attractive targets for modulation giventheir role in the regulation of diverse biological processes. It has nowbeen found that compounds described herein, and pharmaceuticallyacceptable salts and compositions thereof, are effective as inhibitorsof arginine methyltransferases. Such compounds have the general Formula(I):

or a pharmaceutically acceptable salt thereof, wherein R₁, R₃, R_(x), X,Y, and m are as defined herein.

In some embodiments, pharmaceutical compositions are provided whichcomprise a compound described herein (e.g., a compound of Formula (I)),or a pharmaceutically acceptable salt thereof, and optionally apharmaceutically acceptable excipient.

In certain embodiments, compounds described herein inhibit activity ofan arginine methyltransferase (RMT) (e.g., PRMT1, PRMT3, CARM1, PRMT6,and/or PRMT8). In certain embodiments, methods of inhibiting an argininemethyltransferase are provided which comprise contacting the argininemethyltransferase with an effective amount of a compound of Formula (I),or a pharmaceutically acceptable salt thereof. The RMT may be purifiedor crude, and may be present in a cell, tissue, or a subject. Thus, suchmethods encompass inhibition of RMT activity both in vitro and in vivo.In certain embodiments, the RMT is wild-type. In certain embodiments,the RMT is overexpressed. In certain embodiments, the RMT is a mutant.In certain embodiments, the RMT is in a cell. In certain embodiments,the RMT is in an animal, e.g., a human. In some embodiments, the RMT isexpressed at normal levels in a subject, but the subject would benefitfrom RMT inhibition (e.g., because the subject has one or more mutationsin an RMT substrate that causes an increase in methylation of thesubstrate with normal levels of RMT). In some embodiments, the RMT is ina subject known or identified as having abnormal RMT activity (e.g.,overexpression).

In certain embodiments, methods of modulating gene expression in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, cellis in an animal, e.g., a human.

In certain embodiments, methods of modulating transcription in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, thecell is in an animal, e.g., a human.

In some embodiments, methods of treating an RMT-mediated disorder (e.g.,a PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8-mediated disorder) areprovided which comprise administering to a subject suffering from anRMT-mediated disorder an effective amount of a compound described herein(e.g., a compound of Formula (I)), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the RMT-mediated disorder is a proliferative disorder. Incertain embodiments, compounds described herein are useful for treatingcancer. In certain embodiments, compounds described herein are usefulfor treating breast cancer, prostate cancer, lung cancer, colon cancer,bladder cancer, or leukemia. In certain embodiments, the RMT-mediateddisorder is a muscular disorder. In certain embodiments, theRMT-mediated disorder is an autoimmune disorder. In certain embodiments,the RMT-mediated disorder is a neurological disorder. In certainembodiments, the RMT-mediated disorder is a vascular disorder. Incertain embodiments, the RMT-mediated disorder is a metabolic disorder.

Compounds described herein are also useful for the study of argininemethyltransferases in biological and pathological phenomena, the studyof intracellular signal transduction pathways mediated by argininemethyltransferases, and the comparative evaluation of new RMTinhibitors.

This application refers to various issued patent, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosureadditionally encompasses compounds described herein as individualisomers substantially free of other isomers, and alternatively, asmixtures of various isomers.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofany compound described herein does not exclude any tautomer form.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon bya ¹³C- or ¹⁴C-enriched carbon are within the scope of the disclosure.Such compounds are useful, for example, as analytical tools or probes inbiological assays.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. In certain embodiments, each instance of an alkyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group isunsubstituted C₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, thealkyl group is substituted C₁₋₁₀ alkyl.

In some embodiments, an alkyl group is substituted with one or morehalogens. “Perhaloalkyl” is a substituted alkyl group as defined hereinwherein all of the hydrogen atoms are independently replaced by ahalogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, thealkyl moiety has 1 to 8 carbon atoms (“C₁₋₈ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 6 carbon atoms (“C₁₋₆perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 4 carbonatoms (“C₁₋₄ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 3 carbon atoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkylmoiety has 1 to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In someembodiments, all of the hydrogen atoms are replaced with fluoro. In someembodiments, all of the hydrogen atoms are replaced with chloro.Examples of perhaloalkyl groups include —CF₃, —CF₂CF₃, —CF₂CF₂CF₃,—CCl₃, —CFCl₂, —CF₂Cl, and the like.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms and one ormore carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds), andoptionally one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds)(“C₂₋₂₀ alkenyl”). In certain embodiments, alkenyl does not comprisetriple bonds. In some embodiments, an alkenyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkenyl”). In some embodiments, an alkenyl group has 2 to9 carbon atoms (“C₂₋₉ alkenyl”). In some embodiments, an alkenyl grouphas 2 to 8 carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, analkenyl group has 2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In someembodiments, an alkenyl group has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”).In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C₂₋₅alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms(“C₂₋₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon doublebonds can be internal (such as in 2-butenyl) or terminal (such as in1-butenyl). Examples of C₂₋₄ alkenyl groups include ethenyl (C₂),1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄),butadienyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄ alkenyl groups as well as pentenyl (C₅),pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples ofalkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and thelike. In certain embodiments, each instance of an alkenyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) withone or more substituents. In certain embodiments, the alkenyl group isunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis substituted C₂₋₁₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms and one ormore carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds), andoptionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C₂₋₂₀ alkynyl”). In certain embodiments, alkynyl does not comprisedouble bonds. In some embodiments, an alkynyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, analkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In someembodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”).In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C₂₋₅alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms(“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has 2 to 3carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynyl group has2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbon triplebonds can be internal (such as in 2-butynyl) or terminal (such as in1-butynyl). Examples of C₂₋₄ alkynyl groups include, without limitation,ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄),2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl(C₆), and the like. Additional examples of alkynyl include heptynyl(C₇), octynyl (C₈), and the like. In certain embodiments, each instanceof an alkynyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is substituted C₂₋₁₀ alkynyl.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclyl ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. In certain embodiments, each instance of acarbocyclyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). In certain embodiments,each instance of a cycloalkyl group is independently unsubstituted (an“unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”)with one or more substituents. In certain embodiments, the cycloalkylgroup is unsubstituted C₃₋₁₀ cycloalkyl. In certain embodiments, thecycloalkyl group is substituted C₃₋₁₀ cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-10 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or a fused, bridged or spiro ring system such as a bicyclic system(“bicyclic heterocyclyl”), and can be saturated or can be partiallyunsaturated. Heterocyclyl bicyclic ring systems can include one or moreheteroatoms in one or both rings. “Heterocyclyl” also includes ringsystems wherein the heterocyclyl ring, as defined above, is fused withone or more carbocyclyl groups wherein the point of attachment is eitheron the carbocyclyl or heterocyclyl ring, or ring systems wherein theheterocyclyl ring, as defined above, is fused with one or more aryl orheteroaryl groups, wherein the point of attachment is on theheterocyclyl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heterocyclylring system. In certain embodiments, each instance of heterocyclyl isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. Incertain embodiments, the heterocyclyl group is substituted 3-10 memberedheterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiorenyl.Exemplary 4-membered heterocyclyl groups containing one heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing one heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanyl.Exemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl, and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 nelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. In certainembodiments, each instance of an aryl group is independently optionallysubstituted, e.g., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, e.g., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certainembodiments, each instance of a heteroaryl group is independentlyoptionally substituted, e.g., unsubstituted (“unsubstituted heteroaryl”)or substituted (“substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group issubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.Exemplary 5,6-bicyclic heteroaryl groups include, without limitation,any one of the following formulae:

In any of the monocyclic or bicyclic heteroaryl groups, the point ofattachment can be any carbon or nitrogen atom, as valency permits.

“Partially unsaturated” refers to a group that includes at least onedouble or triple bond. The term “partially unsaturated” is intended toencompass rings having multiple sites of unsaturation, but is notintended to include aromatic groups (e.g., aryl or heteroaryl groups) asherein defined. Likewise, “saturated” refers to a group that does notcontain a double or triple bond, i.e., contains all single bonds.

In some embodiments, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl groups, as defined herein, are optionallysubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted”or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat at least one hydrogen present on a group (e.g., a carbon ornitrogen atom) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group has a substituent at one ormore substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, including any of the substituents described hereinthat results in the formation of a stable compound. The presentdisclosure contemplates any and all such combinations in order to arriveat a stable compound. For purposes of this disclosure, heteroatoms suchas nitrogen may have hydrogen substituents and/or any suitablesubstituent as described herein which satisfy the valencies of theheteroatoms and results in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, ortwo R^(aa) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee), —NRCO₂R^(ee),—NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee),OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂,—NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee), SO₂N(R^(ff))₂,—SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —S(═O)R^(ee), —Si(R^(ee))₃,—OSi(R^(ee))₃, —C(═S)N(R^(ff))₂, —C(═O)SR^(ee), —C(═S)SR^(ee),—SC(═S)SR^(ee), —P(═O)₂R^(ee), —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂,—OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(gg) groups, or two geminal R^(dd) substituents canbe joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R^(ff) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₄ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₄ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X is a counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc), and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Amide nitrogen protecting groups (e.g., —C(═O)R^(aa)) include, but arenot limited to, formamide, acetamide, chloroacetamide,trichloroacetamide, trifluoroacetamide, phenylacetamide,3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide,N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide,o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide,(N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine,o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Carbamate nitrogen protecting groups (e.g., —C(═O)OR^(aa)) include, butare not limited to, methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Sulfonamide nitrogen protecting groups (e.g., —S(═O)₂R^(aa)) include,but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, co-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, o-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on a sulfur atom is asulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The present disclosureis not intended to be limited in any manner by the above exemplarylisting of substituents.

“Pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and other animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds describe herein include those derived from suitable inorganicand organic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid, or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, quaternary salts.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (e.g., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example, non-human mammals (e.g., primates (e.g.,cynomolgus monkeys, rhesus monkeys); commercially relevant mammals suchas cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys), rodents (e.g., rats and/or mice), reptiles, amphibians, andfish. In certain embodiments, the non-human animal is a mammal. Thenon-human animal may be a male or female at any stage of development. Anon-human animal may be a transgenic animal.

“Condition,” “disease,” and “disorder” are used interchangeably herein.

“Treat,” “treating” and “treatment” encompasses an action that occurswhile a subject is suffering from a condition which reduces the severityof the condition or retards or slows the progression of the condition(“therapeutic treatment”). “Treat,” “treating” and “treatment” alsoencompasses an action that occurs before a subject begins to suffer fromthe condition and which inhibits or reduces the severity of thecondition (“prophylactic treatment”).

An “effective amount” of a compound refers to an amount sufficient toelicit the desired biological response, e.g., treat the condition. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of a compound described herein may vary depending onsuch factors as the desired biological endpoint, the pharmacokinetics ofthe compound, the condition being treated, the mode of administration,and the age and health of the subject. An effective amount encompassestherapeutic and prophylactic treatment.

A “therapeutically effective amount” of a compound is an amountsufficient to provide a therapeutic benefit in the treatment of acondition or to delay or minimize one or more symptoms associated withthe condition. A therapeutically effective amount of a compound means anamount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of the condition, or enhances the therapeutic efficacy of anothertherapeutic agent.

A “prophylactically effective amount” of a compound is an amountsufficient to prevent a condition, or one or more symptoms associatedwith the condition or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of a therapeutic agent,alone or in combination with other agents, which provides a prophylacticbenefit in the prevention of the condition. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

As used herein, the term “methyltransferase” represents transferaseclass enzymes that are able to transfer a methyl group from a donormolecule to an acceptor molecule, e.g., an amino acid residue of aprotein or a nucleic base of a DNA molecule. Methytransferases typicallyuse a reactive methyl group bound to sulfur in S-adenosyl methionine(SAM) as the methyl donor. In some embodiments, a methyltransferasedescribed herein is a protein methyltransferase. In some embodiments, amethyltransferase described herein is a histone methyltransferase.Histone methyltransferases (HMT) are histone-modifying enzymes,(including histone-lysine N-methyltransferase and histone-arginineN-methyltransferase), that catalyze the transfer of one or more methylgroups to lysine and arginine residues of histone proteins. In certainembodiments, a methyltransferase described herein is a histone-arginineN-methyltransferase.

As generally described above, provided herein are compounds useful asarginine methyltransferase (RMT) inhibitors. In some embodiments, thepresent disclosure provides a compound of Formula (I):

wherein:

X is NR₂ and Y is N; or

X is N and Y is NR₂;

each instance of R₁ is independently selected from the group consistingof hydrogen, halogen, —N₃, —CN, —NO₂, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂;

m is 0, 1, 2, 3, or 4;

R₂ is hydrogen, optionally substituted C₁₋₆ alkyl, optionallysubstituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl,optionally substituted C₃₋₇ cycloalkyl, optionally substituted 4- to7-membered heterocyclyl; or optionally substituted C₁₋₄ alkyl-Cy;

R₃ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄ carbocylyl;

R_(X) is independently optionally substituted C₁₋₄ alkyl or optionallysubstituted C₃₋₄ carbocylyl;

each instance of R^(A) is independently selected from the groupconsisting of hydrogen, optionally substituted acyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, an oxygenprotecting group when attached to an oxygen atom, and a sulfurprotecting group when attached to a sulfur atom;

each instance of R^(B) is independently selected from the groupconsisting of hydrogen, optionally substituted acyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, and a nitrogenprotecting group, or two R^(B) groups are taken together with theirintervening atoms to form an optionally substituted heterocyclic ring;and

each instance of Cy is independently optionally substituted C₃₋₇cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl.

In certain embodiments, a provided compound is of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R₃,R_(x), and R₄ are as described herein.

In certain embodiments, a provided compound is of Formula (II-a):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x)and R₄ are as described herein.

In certain embodiments, a provided compound is of Formula (II-a1):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₄, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a1-i):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, V₁, V₂, V₃, V₄, V₅, p, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a1-ii):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, p, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a1-iii)

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, p, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a1-iv):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, p, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a1-v):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, p, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a2):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, R^(B), q, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a3):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R^(A), and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-b):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₄, and s are as described herein.

In certain embodiments, a provided compound is of Formula (II-b1):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),and R₄ are as described herein.

In certain embodiments, a provided compound is of Formula (II-b2):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),V₁, V₂, V₃, V₄, V₅, R₆, p, and s are as described herein.

In certain embodiments, a provided compound is of Formula (II-b2-i):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₇, p, and s are as described herein.

In certain embodiments, a provided compound is of Formula (II-b3):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, p, and s are as described herein.

In certain embodiments, a provided compound is of Formula (II-c):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₄, t, and R^(B) are as described herein.

In certain embodiments, a provided compound is of Formula (II-c1):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₄, and R^(B) are as described herein.

In certain embodiments, a provided compound is of Formula (II-c2):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, V₁, V₂, V₃, V₄, V₅, R^(B), p, and t are as described herein.

In certain embodiments, a provided compound is of Formula (II-c2-i):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₇, R^(B), p, and t are as described herein.

In certain embodiments, a provided compound is of Formula (II-c3):

or a pharmaceutically acceptable salt thereof, wherein X, Y, R₃, R_(x),R₆, R^(B), q, and t are as described herein.

As defined generally above, each instance of R₁ is independentlyselected from the group consisting of hydrogen, halogen, —N₃, —CN, —NO₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, —OR^(A),—N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),—OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A),—SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In certain embodiments, R₁is hydrogen. In some embodiments, R₁ is not hydrogen. In someembodiments, R₁ is halogen. In certain embodiments, R₁ is F. In certainembodiments, R₁ is Cl. In certain embodiments, R₁ is Br. In certainembodiments, R₁ is I. In some embodiments, R₁ is optionally substitutedalkyl. In some embodiments, R₁ is optionally substituted alkenyl. Incertain embodiments, R₁ is optionally substituted alkynyl. In certainembodiments, R₁ is optionally substituted C₁₋₆ alkyl. In certainembodiments, R₁ is substituted C₁₋₆ alkyl. In certain embodiments, R₁ is—CF₃, CHF₂, or CH₂F. In certain embodiments, R₁ is —C₁₋₆alkyl-carbocyclyl. In certain embodiments, R₁ is —CH₂-cyclopropyl or—CH₂-cyclobutyl. In certain embodiments, R₁ is unsubstituted C₁₋₆ alkyl.In certain embodiments, R₁ is methyl, ethyl, propyl, butyl, or pentyl.In certain embodiments, R₁ is isopropyl, isobutyl, or isopentyl. Incertain embodiments, R₁ is isobutyl. In some embodiments, R₁ is —CN. Insome embodiments, R₁ is optionally substituted carbocyclyl, optionallysubstituted phenyl, optionally substituted heterocyclyl, or optionallysubstituted heteroaryl. In certain embodiments, R₁ is —N(R^(B))₂. Incertain embodiments, R₁ is —NHR^(B). In certain embodiments, R₁ is —NH₂.In certain embodiments, R₁ is —OR^(A). In certain embodiments, R₁ is—OH. In certain embodiments, R₁ is —OR^(A), wherein R^(A) is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl. In certain embodiments, R₁ is —O-isobutylenyl. Incertain embodiments, R₁ is —OR^(A), wherein R^(A) is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, R₁ is —OR^(A), whereinR^(A) is unsubstituted C₁₋₆ alkyl. In certain embodiments, R₁ is—O-propyl, —O-isopropyl, —O-isobutyl, or —O-isopentyl. In certainembodiments, R₁ is —OR^(A), wherein R^(A) is substituted C₁₋₆ alkyl. Incertain embodiments, R₁ is —O—C₁₋₆alkyl-O—C₁₋₆alkyl. In certainembodiments, R₁ is —OCH₂CH₂OCH₃ or —OCH₂CH₂CH₂OCH₃. In certainembodiments, R₁ is —O—C₁₋₆alkyl-carbocyclyl. In certain embodiments, R₁is —O—CH₂-cyclobutyl or —O—CH₂-cyclopropyl. In certain embodiments, R₁is —O—C₁₋₆alkyl-heterocyclyl. In certain embodiments, R₁ is—O—CH₂-tetrahydropyranyl or —O—CH₂-oxetanyl. In certain embodiments, R₁is —OR^(A), wherein R^(A) is optionally substituted heterocyclyl. Incertain embodiments, R₁ is —O— tetrahydropyranyl or —O-oxetanyl. Incertain embodiments, R₁ is —OR^(A), wherein R^(A) is optionallysubstituted aryl. In certain embodiments, R₁ is —O-phenyl. In certainembodiments, R₁ is —OR^(A), wherein R^(A) is optionally substitutedheteroaryl.

In certain embodiments, R₁ is —Z—R₄, wherein Z is a bond, —O—, —S—,—NR^(B)—, —NR^(B)C(═O)—, —C(═O)NR^(B)—, —OC(═O)NR^(B)—, —NR^(B)C(═O)O—,—SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, trans-CR^(C)═CR^(C)—,cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—, —C(R^(C))₂O—, —S(═O)₂O—,—OS(═O)₂—, —S(═O)₂NR^(B)—, —NR^(B)S(═O)₂—, or an optionally substitutedC₁₋₈ hydrocarbon chain, optionally wherein one or more carbon units ofthe hydrocarbon chain is replaced with —O—, —S—, —NR^(B)—,—NR^(B)C(═O)—, —C(═O)NR^(B)—, —OC(═O)NR^(B)—, —NR^(B)C(═O)O—, —SC(═O)—,—C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^(B)C(═S)—, —C(═S)NR^(B)—,trans-CR^(C)═CR^(C)—, cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—,—C(R^(C))₂O—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, —NR^(B)S(═O)₂—,wherein each instance of R^(C) is independently selected from the groupconsisting of hydrogen, halogen optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted carbocyclyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheterocyclyl, and optionally substituted heteroaryl, or two R^(C) groupsare joined to form an optionally substituted carbocyclic or optionallysubstituted heterocyclic ring; and R₄ is hydrogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), or an oxygen protecting group.In certain embodiments, Z is —O—. In certain embodiments, Z isoptionally substituted C₁₋₈ hydrocarbon chain, optionally wherein one ormore carbon units of the hydrocarbon chain is replaced with —O—, —S—,—NR^(B)—, —NR^(B)C(═O)—, —C(═O)NR^(B)—, —OC(═O)NR^(B)—, —NR^(B)C(═O)O—,—SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^(B)C(═S)—, —C(═S)NR^(B)—,trans-CR^(C)═CR^(C)—, cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—,—C(R^(C))₂O—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, or —NR^(B)S(═O)₂—.In certain embodiments, Z is —(CH₂)_(n)O—, wherein n is 1, 2, 3, 4, 5,6, 7, or 8. In certain embodiments, Z is —CH₂O—. In certain embodiments,Z is —(CH₂)₂O—. In certain embodiments, Z is —(CH₂)₃O—. In certainembodiments, Z is —(CH₂)₄O—. In certain embodiments, Z is —(CH₂)₅O—. Incertain embodiments, Z is —(CH₂)₆O—. In certain embodiments, Z is—(CH₂)₇O—. In certain embodiments, Z is —(CH₂)₈O—. In certainembodiments, Z is optionally substituted alkylene. In certainembodiments, Z is —(CH₂)_(s)—, wherein s is 1, 2, 3, 4, 5, 6, 7, or 8.In certain embodiments, Z is —CH₂—. In certain embodiments, Z is—(CH₂)₂—. In certain embodiments, Z is —(CH₂)₃—. In certain embodiments,Z is —(CH₂)₄—. In certain embodiments, Z is —(CH₂)₅—. In certainembodiments, Z is —(CH₂)₆—. In certain embodiments, Z is —(CH₂)₇—. Incertain embodiments, Z is —(CH₂)₈—. In certain embodiments, Z is—(CH₂)_(t)—C(═O)NR^(B)—, wherein t is 0, 1, 2, 3, 4, 5, 6, 7, or 8. Incertain embodiments, Z is —C(═O)NR^(B)—. In certain embodiments, Z is—CH₂—C(═O)NR^(B)—. In certain embodiments, Z is —(CH₂)₂—C(═O)NR^(B)—. Incertain embodiments, Z is —(CH₂)₃—C(═O)NR^(B)—. In certain embodiments,Z is —(CH₂)₄—C(═O)NR^(B)—. In certain embodiments, Z is—(CH₂)₅—C(═O)NR^(B)—. In certain embodiments, Z is —(CH₂)₆—C(═O)NR^(B)—.In certain embodiments, Z is —(CH₂)₇—C(═O)NR^(B)—. In certainembodiments, Z is —(CH₂)₈—C(═O)NR^(B)—. In certain embodiments, Z is—NR^(B)C(═O)—. In certain embodiments, Z is —S—. In certain embodiments,Z is —NR^(B)—. In certain embodiments, Z is —OC(═O)NR^(B)—. In certainembodiments, Z is —NR^(B)C(═O)O—. In certain embodiments, Z is —SC(═O)—.In certain embodiments, Z is —C(═O)S—. In certain embodiments, Z is—OC(═O)—. In certain embodiments, Z is —C(═O)O—. In certain embodiments,Z is —NR^(B)C(═S)—. In certain embodiments, Z is —C(═S)NR^(B)—. Incertain embodiments, Z is trans-CR^(C)═CR^(C)—. In certain embodiments,Z is cis-CR^(C)═CR^(C)—. In certain embodiments, Z is —C≡C—. In certainembodiments, Z is —OC(R_(C))₂—. In certain embodiments, Z is —S(═O)₂O—.In certain embodiments, Z is —OS(═O)₂—. In certain embodiments, Z is—OS(═O)₂—. In certain embodiments, Z is —S(═O)₂NR^(B)—. In certainembodiments, Z is —NR^(B)S(═O)₂—. In certain embodiments, R₄ ishydrogen. In some embodiments, R₄ is optionally substituted alkyl. Insome embodiments, R₄ is optionally substituted alkenyl. In certainembodiments, R₄ is optionally substituted alkynyl. In certainembodiments, R₄ is optionally substituted C₁₋₆ alkyl. In certainembodiments, R₄ is substituted C₁₋₆ alkyl. In certain embodiments, R₄ isunsubstituted C₁₋₆ alkyl. In certain embodiments, R₄ is methyl, ethyl,propyl, butyl, pentyl, isopropyl, isobutyl, or isopentyl. In certainembodiments, R₄ is optionally substituted carbocyclyl. In someembodiments, R₄ is optionally substituted cyclocyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. In certain embodiments, R₄ is of the formula

wherein R₅ is optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl. In certain embodiments,R₅ is optionally substituted methyl, ethyl, propyl, butyl, pentyl,isopropyl, isobutyl, or isopentyl. In certain embodiments, R₅ is benzyl.In certain embodiments, R₅ is methyl, ethyl, propyl, butyl, pentyl,isopropyl, isobutyl, or isopentyl. In certain embodiments, R₅ isisopropyl. In certain embodiments, R₅ is optionally substituted aryl. Incertain embodiments, R₅ is phenyl. In certain embodiments, R₄ isoptionally substituted aryl. In certain embodiments, R₄ is optionallysubstituted heterocyclyl. In certain embodiments, R₄ is optionallysubstituted five-membered heterocyclyl. In certain embodiments, R₄ isoptionally substituted six-membered heterocyclyl. In certainembodiments, R₄ is optionally substituted five-membered heteroaryl. Incertain embodiments, R₄ is optionally substituted pyrrole, furan,thiophene, imidazole, pyrazole, isothiazole isoxazole, oxazole,thiazole, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-oxadiazole,1,2,4-thiadiazole, 1,2,5-oxadiazole, 1,2,5-thiadiazole, 1,2,3-triazole,1,2,4-triazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole,1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, or 2H-tetrazole. In certainembodiments, R₄ is optionally substituted six-membered heteroaryl. Incertain embodiments, R₄ is optionally substituted six-memberedheteroaryl. In certain embodiments, R₄ is optionally substitutedpyridine. In certain embodiments, R₄ is —OR^(A). In certain embodiments,R₄ is —OH. In certain embodiments, R₄ is —O-methyl, —O-ethyl, —O-propyl,—O-butyl, —O-isopropyl, or —O-isobutyl.

In certain embodiments, Z is —O— and R₄ is optionally substituted alkyl.In certain embodiments, Z is —O— and R₄ is optionally substitutedcarbocyclyl. In certain embodiments, Z is —O— and R₄ is of the formula

In certain embodiments, Z is —(CH₂)_(n)O— and R₄ is optionallysubstituted alkyl. In certain embodiments, Z is —(CH₂)_(n)O— and R₄ is—OR^(A). In certain embodiments, Z is —(CH₂)_(n)O— and R₄ is —N(R^(B))₂.In certain embodiments, Z is —(CH₂)_(n)O— and R₄ is —SR^(A). In certainembodiments, Z is —(CH₂)_(n)O— and R₄ is optionally substitutedsix-membered heteroaryl. In certain embodiments, Z is —(CH₂)_(n)O— andR₄ is optionally substituted five-membered heteroaryl. In certainembodiments, Z is —(CH₂)_(n)O— and R₄ is optionally substitutedsix-membered heteroaryl. In certain embodiments, Z is —(CH₂)_(n)O— andR₄ is optionally substituted pyrazole. In certain embodiments, Z is—(CH₂)_(s)— and R₄ is optionally substituted pyridine. In certainembodiments, Z is —(CH₂)_(s)— and R₄ is optionally substitutedsix-membered heteroaryl. In certain embodiments, Z is —(CH₂)_(s)— and R₄is optionally substituted five-membered heteroaryl. In certainembodiments, Z is —(CH₂)_(s)— and R₄ is optionally substitutedsix-membered heteroaryl. In certain embodiments, Z is —(CH₂)_(s)— and R₄is optionally substituted pyrazole. In certain embodiments, Z is—(CH₂)_(s)— and R₄ is optionally substituted pyridine. In certainembodiments, Z is —(CH₂)_(t)—C(═O)NR^(B)— and R₄ is optionallysubstituted six-membered heteroaryl. In certain embodiments, Z is—(CH₂)_(t)—C(═O)NR^(B)— and R₄ is optionally substituted five-memberedheteroaryl. In certain embodiments, Z is —(CH₂)_(t)—C(═O)NR^(B)— and R₄is optionally substituted six-membered heteroaryl. In certainembodiments, Z is —(CH₂)_(t)—C(═O)NR^(B)— and R₄ is optionallysubstituted pyrazole. In certain embodiments, Z is—(CH₂)_(t)—C(═O)NR^(B)— and R₄ is optionally substituted pyridine.

In certain embodiments, R₄ is of the formula

wherein each of V₁, V₂, V₃, V₄, and V₅ is independently C or N. Incertain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is selected from the group consisting of

As used herein, p is 0, 1, 2, 3, 4, or 5 as valence permits. In someembodiments, p is 0. In some embodiments, p is 1. In some embodiments, pis 2. In some embodiments, p is 3. In some embodiments, p is 4. In someembodiments, p is 5. In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

In certain embodiments, R₄ is of the formula

As used herein, q is 0, 1, or 2. In certain embodiments, q is 1. Incertain embodiments, q is 2.

As described herein, each instance of R₆ is independently selected fromthe group consisting of hydrogen, halogen, —N₃, —CN, —NO₂, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, —OR^(A),—N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),—OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A),—SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In certain embodiments, R₆is hydrogen. In some embodiments, R₆ is not hydrogen. In someembodiments, R₆ is halogen. In certain embodiments, R₆ is F. In certainembodiments, R₆ is Cl. In certain embodiments, R₆ is Br. In certainembodiments, R₆ is I. In some embodiments, R₆ is optionally substitutedalkyl. In some embodiments, R₆ is optionally substituted alkenyl. Incertain embodiments, R₆ is optionally substituted alkynyl. In certainembodiments, R₆ is optionally substituted C₁₋₆ alkyl. In certainembodiments, R₆ is substituted C₁₋₆ alkyl. In certain embodiments, R₆ is—CF₃, CHF₂, or CH₂F. In certain embodiments, R₆ is —C₁₋₆alkyl-carbocyclyl. In certain embodiments, R₆ is —CH₂-cyclopropyl or—CH₂-cyclobutyl. In certain embodiments, R₆ is unsubstituted C₁₋₆ alkyl.In certain embodiments, R₆ is methyl, ethyl, propyl, butyl, or pentyl.In certain embodiments, R₆ is isopropyl, isobutyl, or isopentyl. Incertain embodiments, R₁ is isobutyl. In some embodiments, R₆ is —CN. Insome embodiments, R₆ is optionally substituted carbocyclyl, optionallysubstituted phenyl, optionally substituted heterocyclyl, or optionallysubstituted heteroaryl. In certain embodiments, R₆ is —N(R^(B))₂. Incertain embodiments, R₆ is —NHR^(B). In certain embodiments, R₁ is —NH₂.In certain embodiments, R₆ is —OR^(A). In certain embodiments, R₆ is—OH. In certain embodiments, R₆ is —OR^(A), wherein R^(A) is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl. In certain embodiments, R₆ is —O-isobutylenyl. Incertain embodiments, R₆ is —OR^(A), wherein R^(A) is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, R₆ is —OR^(A), whereinR^(A) is unsubstituted C₁₋₆ alkyl. In certain embodiments, R₆ is—O-methyl, —O-ethyl, —O-propyl, —O-butyl, —O-pentyl, —O-isopropyl,—O-isobutyl, or —O— isopentyl.

As defined generally above, R₂ is hydrogen, optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₇ cycloalkyl, optionally substituted4- to 7-membered heterocyclyl; or optionally substituted C₁₋₄ alkyl-Cy,wherein Cy is optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,or optionally substituted heteroaryl. As defined generally above, eachinstance of Cy is independently optionally substituted C₃₋₇ cycloalkyl,optionally substituted 4- to 7-membered heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl. In someembodiments, Cy is optionally substituted C₃₋₇ cycloalkyl. In someembodiments, Cy is optionally substituted 4- to 7-membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, andsulfur. In some embodiments, Cy is oxetane, tetrahydrofuran, ortetrahydropyran. In some embodiments, Cy is optionally substituted aryl.In some embodiments, Cy is optionally substituted phenyl. In someembodiments, Cy is unsubstituted phenyl. In some embodiments, Cy isoptionally substituted heteroaryl having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, Cy isoptionally substituted 5- to 6-membered heteroaryl having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, Cy is pyridyl. In some embodiments, R₂ is

In some embodiments, R₂ is

In some embodiments, R₂ is

As defined generally above, R₃ is hydrogen, C₁₋₄ alkyl, or C₃₋₄cycloalkyl. In certain embodiments, R₃ is hydrogen. In certainembodiments, R₃ is C₁₋₄ alkyl. In certain embodiments, R₃ is methyl,ethyl, propyl, butyl, or pentyl. In certain embodiments, R₃ isisopropyl, isobutyl, or isopentyl. In certain embodiments, R₃ isisobutyl. In certain embodiments, R³ is C₃₋₄ cycloalkyl. In certainembodiments, R³ is cyclopropyl. In certain embodiments, R³ iscyclobutyl.

As defined generally above, R_(x) is optionally substituted C₁₋₄ alkylor optionally substituted C₃₋₄ cycloalkyl. In certain embodiments, R_(x)is unsubstituted C₁₋₄ alkyl. In certain embodiments, R_(x) is methyl. Incertain embodiments, R_(x) is ethyl. In certain embodiments, R_(x) isisopropyl. In certain embodiments, R_(x) is propyl. In certainembodiments, R_(x) is butyl. In certain embodiments, R_(x) issubstituted C₁₋₄ alkyl. In certain embodiments, R_(x) is C₁₋₄ alkylsubstituted with hydroxyl or alkoxy. In certain embodiments, R_(x) ishydroxyethyl or methoxyethyl. In certain embodiments, R_(x) isoptionally substituted C₃₋₄ cycloalkyl. In certain embodiments, R_(x) isunsubstituted C₃₋₄ cycloalkyl. In certain embodiments, R_(x) issubstituted cyclopropyl. In certain embodiments, R_(x) is unsubstitutedcyclopropyl. In certain embodiments, R_(x) is substituted cyclobutyl. Incertain embodiments, R_(x) is unsubstituted cyclobutyl.

As defined generally above, m is 0, 1, 2, 3, or 4. In certainembodiments, m is 0. In certain embodiments, m is 1. In certainembodiments, m is 2. In certain embodiments, m is 3. In certainembodiments, m is 4.

As defined generally above, each instance of R^(A) is independentlyselected from the group consisting of hydrogen, optionally substitutedacyl, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted alkyl-Cy, anoxygen protecting group when attached to an oxygen atom, and a sulfurprotecting group when attached to a sulfur atom. In certain embodiments,R^(A) is hydrogen. In certain embodiments, R^(A) is optionallysubstituted acyl. In certain embodiments, R^(A) is optionallysubstituted alkyl. In certain embodiments, R^(A) is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, R^(A) is substitutedC₁₋₆ alkyl. In certain embodiments, R^(A) is unsubstituted C₁₋₆ alkyl.In certain embodiments, R^(A) is methyl, ethyl, propyl, butyl, pentyl,isopropyl, isobutyl, or isopentyl. In certain embodiments, R^(A) isoptionally substituted alkenyl. In certain embodiments, R^(A) isoptionally substituted alkynyl. In certain embodiments, R^(A) isoptionally substituted carbocyclyl. In certain embodiments, R^(A) isoptionally substituted aryl. In certain embodiments, R^(A) is optionallysubstituted heterocyclyl. In certain embodiments, R^(A) is optionallysubstituted heteroaryl. In certain embodiments, R^(A) is optionallysubstituted alkyl-Cy. In certain embodiments, R^(A) is an oxygenprotecting group. In certain embodiments, R^(A) is a sulfur protectinggroup.

As defined generally above, each instance of R^(B) is independentlyselected from the group consisting of hydrogen, optionally substitutedacyl, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted alkyl-Cy, anda nitrogen protecting group, or two R^(B) groups are taken together withtheir intervening atoms to form an optionally substituted heterocyclicring. In certain embodiments, R^(B) is hydrogen. In certain embodiments,R^(B) is optionally substituted acyl. In certain embodiments, R^(B) isoptionally substituted alkyl. In certain embodiments, R^(B) isoptionally substituted C₁₋₆ alkyl. In certain embodiments, R^(B) issubstituted C₁₋₆ alkyl. In certain embodiments, R^(B) is unsubstitutedC₁₋₆ alkyl. In certain embodiments, R^(B) is methyl, ethyl, propyl,butyl, pentyl, isopropyl, isobutyl, or isopentyl. In certainembodiments, R^(B) is optionally substituted alkenyl. In certainembodiments, R^(B) is optionally substituted alkynyl. In certainembodiments, R^(B) is optionally substituted carbocyclyl. In certainembodiments, R^(B) is optionally substituted aryl. In certainembodiments, R^(B) is optionally substituted heterocyclyl. In certainembodiments, R^(B) is optionally substituted heteroaryl. In certainembodiments, R^(B) is optionally substituted alkyl-Cy. In certainembodiments, R^(B) is a nitrogen protecting group. In certainembodiments, two R^(B) groups are taken together with their interveningatoms to form an optionally substituted heterocyclic ring.

In certain embodiments, a provided compound is a compound listed inTable 1, or a pharmaceutically acceptable salt thereof.

TABLE 1 Exemplary Compounds Cmpd LC-MS m/z No Structure (M + H) 1

311.2 2

277.1 3

305.1 4

305.1 5

293.0 6

307.1 7

339.1 8

305.1 9

353.1 10 

311.1 11 

303.1 12 

326.1 13 

350.1 14 

350.1 15 

326.1 16 

326.1 17 

315.1

In certain embodiments, a provided compound inhibits an RMT (e.g.,PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, aprovided compound inhibits wild-type PRMT1, PRMT3, CARM1, PRMT6, and/orPRMT8. In certain embodiments, a provided compound inhibits a mutantRMT. In certain embodiments, a provided compound inhibits PRMT1, PRMT3,CARM1, PRMT6, and/or PRMT8, e.g., as measured in an assay describedherein. In certain embodiments, the RMT is from a human. In certainembodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3,CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than or equal to 10 μM. Incertain embodiments, a provided compound inhibits an RMT (e.g., PRMT1,PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than or equal to 1μM. In certain embodiments, a provided compound inhibits an RMT (e.g.,PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than or equalto 0.1 μM. In certain embodiments, a provided compound inhibits an RMT(e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than orequal to 0.01 μM. In certain embodiments, a provided compound inhibitsan RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at anEC₃₀ less than or equal to 10 μM. In certain embodiments, a providedcompound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/orPRMT8) in a cell at an EC₃₀ less than or equal to 12 μM. In certainembodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3,CARM1, PRMT6, and/or PRMT8) in a cell at an EC₃₀ less than or equal to 3μM. In certain embodiments, a provided compound inhibits PRMT1 in a cellat an EC₃₀ less than or equal to 12 μM. In certain embodiments, aprovided compound inhibits PRMT1 in a cell at an EC₃₀ less than or equalto 3 μM. In certain embodiments, a provided compound inhibits an RMT(e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC₃₀less than or equal to 1 μM. In certain embodiments, a provided compoundinhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in acell at an EC₃₀ less than or equal to 0.1 μM. In certain embodiments, aprovided compound inhibits cell proliferation at an EC₅₀ less than orequal to 10 μM. In certain embodiments, a provided compound inhibitscell proliferation at an EC₅₀ less than or equal to 1 μM. In certainembodiments, a provided compound inhibits cell proliferation at an EC₅₀less than or equal to 0.1 μM.

It will be understood by one of ordinary skill in the art that the RMTcan be wild-type, or any mutant or variant.

The present disclosure provides pharmaceutical compositions comprising acompound described herein, e.g., a compound of Formula (I), or apharmaceutically acceptable salt thereof, as described herein, andoptionally a pharmaceutically acceptable excipient. It will beunderstood by one of ordinary skill in the art that the compoundsdescribed herein, or salts thereof, may be present in various forms,such as amorphous, hydrates, solvates, or polymorphs. In certainembodiments, a provided composition comprises two or more compoundsdescribed herein. In certain embodiments, a compound described herein,or a pharmaceutically acceptable salt thereof, is provided in aneffective amount in the pharmaceutical composition. In certainembodiments, the effective amount is a therapeutically effective amount.In certain embodiments, the effective amount is an amount effective forinhibiting an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). Incertain embodiments, the effective amount is an amount effective fortreating an RMT-mediated disorder (e.g., a PRMT1, PRMT3, CARM1, PRMT6,and/or PRMT8-mediated disorder). In certain embodiments, the effectiveamount is a prophylactically effective amount. In certain embodiments,the effective amount is an amount effective to prevent an RMT-mediateddisorder.

Pharmaceutically acceptable excipients include any and all solvents,diluents, or other liquid vehicles, dispersions, suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants, and the like, assuited to the particular dosage form desired. General considerations informulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing a compound described herein (the“active ingredient”) into association with a carrier and/or one or moreother accessory ingredients, and then, if necessary and/or desirable,shaping and/or packaging the product into a desired single- ormulti-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the present disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

In some embodiments, a pharmaceutical composition described herein issterilized.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays(e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminumsilicate)), long chain amino acid derivatives, high molecular weightalcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60),polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate(Span 40), sorbitan monostearate (Span 60], sorbitan tristearate (Span65), glyceryl monooleate, sorbitan monooleate (Span 80)),polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45),polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor™),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimoniumbromide, cetylpyridinium chloride, benzalkonium chloride, docusatesodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(Veegum), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol. Exemplary acidic preservatives include vitaminA, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid,dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixturesthereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the compoundsdescribed herein are mixed with solubilizing agents such as Cremophor™,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the compounds describedherein with suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active ingredient can be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets, and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a providedcompound may include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants and/or patches. Generally, the activeingredient is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and/or any desired preservatives and/or buffers ascan be required. Additionally, the present disclosure encompasses theuse of transdermal patches, which often have the added advantage ofproviding controlled delivery of an active ingredient to the body. Suchdosage forms can be prepared, for example, by dissolving and/ordispensing the active ingredient in the proper medium. Alternatively oradditionally, the rate can be controlled by either providing a ratecontrolling membrane and/or by dispersing the active ingredient in apolymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide the active ingredient in the form of droplets of a solutionand/or suspension. Such formulations can be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising the active ingredient, and mayconveniently be administered using any nebulization and/or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, and/or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 to 500 micrometers. Such a formulation is administered byrapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations for nasal administration may, for example, comprise fromabout as little as 0.1% (w/w) and as much as 100% (w/w) of the activeingredient, and may comprise one or more of the additional ingredientsdescribed herein. A provided pharmaceutical composition can be prepared,packaged, and/or sold in a formulation for buccal administration. Suchformulations may, for example, be in the form of tablets and/or lozengesmade using conventional methods, and may contain, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable and/or degradable composition and, optionally, one or moreof the additional ingredients described herein. Alternately,formulations for buccal administration may comprise a powder and/or anaerosolized and/or atomized solution and/or suspension comprising theactive ingredient. Such powdered, aerosolized, and/or aerosolizedformulations, when dispersed, may have an average particle and/ordroplet size in the range from about 0.1 to about 200 nanometers, andmay further comprise one or more of the additional ingredients describedherein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid carrier. Such drops may further comprisebuffering agents, salts, and/or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis disclosure.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

Compounds provided herein are typically formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of provided compositionswill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular subject or organism will depend upon a variety of factorsincluding the disease, disorder, or condition being treated and theseverity of the disorder; the activity of the specific active ingredientemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific activeingredient employed; the duration of the treatment; drugs used incombination or coincidental with the specific active ingredientemployed; and like factors well known in the medical arts.

The compounds and compositions provided herein can be administered byany route, including enteral (e.g., oral), parenteral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),and/or the condition of the subject (e.g., whether the subject is ableto tolerate oral administration).

The exact amount of a compound required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular compound(s), mode ofadministration, and the like. The desired dosage can be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a compound foradministration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosageform.

In certain embodiments, a compound described herein may be administeredat dosage levels sufficient to deliver from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about mg/kg, from about 0.1 mg/kgto about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 25 mg/kg, of subject body weight per day,one or more times a day, to obtain the desired therapeutic effect.

In some embodiments, a compound described herein is administered one ormore times per day, for multiple days. In some embodiments, the dosingregimen is continued for days, weeks, months, or years.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

It will be also appreciated that a compound or composition, as describedherein, can be administered in combination with one or more additionaltherapeutically active agents. In certain embodiments, a compound orcomposition provided herein is administered in combination with one ormore additional therapeutically active agents that improve itsbioavailability, reduce and/or modify its metabolism, inhibit itsexcretion, and/or modify its distribution within the body. It will alsobe appreciated that the therapy employed may achieve a desired effectfor the same disorder, and/or it may achieve different effects.

The compound or composition can be administered concurrently with, priorto, or subsequent to, one or more additional therapeutically activeagents. In certain embodiments, the additional therapeutically activeagent is a compound of Formula (I). In certain embodiments, theadditional therapeutically active agent is not a compound of Formula(I). In general, each agent will be administered at a dose and/or on atime schedule determined for that agent. In will further be appreciatedthat the additional therapeutically active agent utilized in thiscombination can be administered together in a single composition oradministered separately in different compositions. The particularcombination to employ in a regimen will take into account compatibilityof a provided compound with the additional therapeutically active agentand/or the desired therapeutic effect to be achieved. In general, it isexpected that additional therapeutically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

Exemplary additional therapeutically active agents include, but are notlimited to, small organic molecules such as drug compounds (e.g.,compounds approved by the U.S. Food and Drug Administration as providedin the Code of Federal Regulations (CFR)), peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins, and cells. Incertain embodiments, an additional therapeutically active agent isprednisolone, dexamethasone, doxorubicin, vincristine, mafosfamide,cisplatin, carboplatin, Ara-C, rituximab, azacitadine, panobinostat,vorinostat, everolimus, rapamycin, ATRA (all-trans retinoic acid),daunorubicin, decitabine, Vidaza, mitoxantrone, or IBET-151.

Also encompassed by the present disclosure are kits (e.g.,pharmaceutical packs). The kits provided may comprise a providedpharmaceutical composition or compound and a container (e.g., a vial,ampule, bottle, syringe, and/or dispenser package, or other suitablecontainer). In some embodiments, provided kits may optionally furtherinclude a second container comprising a pharmaceutical excipient fordilution or suspension of a provided pharmaceutical composition orcompound. In some embodiments, a provided pharmaceutical composition orcompound provided in the container and the second container are combinedto form one unit dosage form. In some embodiments, a provided kitsfurther includes instructions for use.

Compounds and compositions described herein are generally useful for theinhibition of RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). Insome embodiments, methods of treating an RMT-mediated disorder in asubject are provided which comprise administering an effective amount ofa compound described herein (e.g., a compound of Formula (I)), or apharmaceutically acceptable salt thereof), to a subject in need oftreatment. In certain embodiments, the effective amount is atherapeutically effective amount. In certain embodiments, the effectiveamount is a prophylactically effective amount. In certain embodiments,the subject is suffering from a RMT-mediated disorder. In certainembodiments, the subject is susceptible to a RMT-mediated disorder.

As used herein, the term “RMT-mediated disorder” means any disease,disorder, or other pathological condition in which an RMT (e.g., PRMT1,PRMT3, CARM1, PRMT6, and/or PRMT8) is known to play a role. Accordingly,in some embodiments, the present disclosure relates to treating orlessening the severity of one or more diseases in which an RMT is knownto play a role.

In some embodiments, the present disclosure provides a method ofinhibiting an RMT comprising contacting the RMT with an effective amountof a compound described herein (e.g., a compound of Formula (I)), or apharmaceutically acceptable salt thereof. The RMT may be purified orcrude, and may be present in a cell, tissue, or subject. Thus, suchmethods encompass both inhibition of in vitro and in vivo RMT activity.In certain embodiments, the method is an in vitro method, e.g., such asan assay method. It will be understood by one of ordinary skill in theart that inhibition of an RMT does not necessarily require that all ofthe RMT be occupied by an inhibitor at once. Exemplary levels ofinhibition of an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8)include at least 10% inhibition, about 10% to about 25% inhibition,about 25% to about 50% inhibition, about 50% to about 75% inhibition, atleast 50% inhibition, at least 75% inhibition, about 80% inhibition,about 90% inhibition, and greater than 90% inhibition.

In some embodiments, provided is a method of inhibiting RMT activity ina subject in need thereof (e.g., a subject diagnosed as having anRMT-mediated disorder) comprising administering to the subject aneffective amount of a compound described herein (e.g., a compound ofFormula (I)), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof.

In certain embodiments, provided is a method of modulating geneexpression in a cell which comprises contacting a cell with an effectiveamount of a compound of Formula (I), or a pharmaceutically acceptablesalt thereof. In certain embodiments, the cell in culture in vitro. Incertain embodiments, the cell is in an animal, e.g., a human. In certainembodiments, the cell is in a subject in need of treatment.

In certain embodiments, provided is a method of modulating transcriptionin a cell which comprises contacting a cell with an effective amount ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof. In certain embodiments, the cell in culture in vitro. Incertain embodiments, the cell is in an animal, e.g., a human. In certainembodiments, the cell is in a subject in need of treatment.

In certain embodiments, a method is provided of selecting a therapy fora subject having a disease associated with an RMT-mediated disorder ormutation comprising the steps of determining the presence of anRMT-mediated disorder or gene mutation in an RMT gene (e.g., a PRMT1,PRMT3, CARM1, PRMT6, and/or PRMT8 gene) or and selecting, based on thepresence of an RMT-mediated disorder a gene mutation in the RMT gene atherapy that includes the administration of a provided compound. Incertain embodiments, the disease is cancer.

In certain embodiments, a method of treatment is provided for a subjectin need thereof comprising the steps of determining the presence of anRMT-mediated disorder or a gene mutation in the RMT gene and treatingthe subject in need thereof, based on the presence of a RMT-mediateddisorder or gene mutation in the RMT gene with a therapy that includesthe administration of a provided compound. In certain embodiments, thesubject is a cancer patient.

In some embodiments, a compound provided herein is useful in treating aproliferative disorder, such as cancer. For example, while not beingbound to any particular mechanism, protein arginine methylation by PRMTsis a modification that has been implicated in signal transduction, genetranscription, DNA repair and mRNA splicing, among others; andoverexpression of PRMTs within these pathways is often associated withvarious cancers. Thus, compounds which inhibit the action of PRMTs, asprovided herein, are effective in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT1. For example, PRMT1overexpression has been observed in various human cancers, including,but not limited to, breast cancer, prostate cancer, lung cancer, coloncancer, bladder cancer, and leukemia. In one example, PRMT1 specificallydeposits an asymmetric dimethylarginine (aDMA) mark on histone H4 atarginine 3 (H4R3me2a), and this mark is associated with transcriptionactivation. In prostate cancer, the methylation status of H4R3positively correlates with increasing tumor grade and can be used topredict the risk of prostate cancer recurrence (Seligson et al., Nature2005 435, 1262-1266). Thus, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with themethylation status of H4R3, e.g., prostate cancer. Additionally, themethylarginine effector molecule TDRD3 interacts with the H4R3me2a mark,and overexpression of TDRD3 is linked to poor prognosis for the survivalof patients with breast cancer (Nagahata et al., Cancer Sci. 2004 95,218-225). Thus, in some embodiments, inhibitors of PRMT1, as describedherein, are useful in treating cancers associated with overexpression ofTDRD3, e.g., breast cancer, as inhibition of PRMT1 leads to a decreasein methylation of H4R3, thereby preventing the association ofoverexpressed TDRD3 with H4R3me2a. In other examples, PRMT1 is known tohave non-histone substrates. For example, PRMT1, when localized to thecytoplasm, methylates proteins that are involved in signal transductionpathways, e.g., the estrogen receptor (ER). The expression status of ERin breast cancer is critical for prognosis of the disease, and bothgenomic and non-genomic ER pathways have been implicated in thepathogenesis of breast cancer. For example, it has been shown that PRMT1methylates ERα, and that ERα methylation is required for the assembly ofERα with SRC (a proto-oncogene tyrosine-protein kinase) and focaladhesion kinase (FAK). Further, the silencing of endogenous PRMT1resulted in the inability of estrogen to activate AKT. These resultssuggested that PRMT1-mediated ERα methylation is required for theactivation of the SRC—PI3K-FAK cascade and AKT, coordinating cellproliferation and survival. Thus, hypermethylation of ERα in breastcancer is thought to cause hyperactivation of this signaling pathway,providing a selective survival advantage to tumor cells (Le Romancer etal., Mol. Cell 2008 31, 212-221; Le Romancer et al., Steroids 2010 75,560-564). Accordingly, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with ERαmethylation, e.g., breast cancer. In yet another example, PRMT1 has beenshown to be involved in the regulation of leukemia development. Forexample, SRC-associated in mitosis 68 kDa protein (SAM68; also known asKHDRBS 1) is a well-characterized PRMT1 substrate, and when either SAM68or PRMT1 is fused directly to the myeloid/lymphoid leukemia (MLL) gene,these fusion proteins can activate MLL oncogenic properties, implyingthat the methylation of SAM68 by PRMT1 is a critical signal for thedevelopment of leukemia (Cheung et al., Nature Cell Biol. 2007 9,1208-1215). Accordingly, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with SAM68methylation, e.g., leukemia. In still another example, PRMT1 isimplicated in leukemia development through its interaction with AE9a, asplice isoform of AMLI-ETO (Shia et al., Blood 2012 119:4953-62).Knockdown of PRMT1 affects expression of certain AE9a-activated genesand suppresses AE9a's self-renewal capability. It has also been shownthat AE9a recruits PRMT1 to AE9a activated gene promoters, which leadsto increased H4 Arg3 methylation, H3 Lys9/14 acetylation, andtranscription activated. Accordingly, in some embodiments, inhibitors ofPRMT1, as described herein, are useful in treating cancers associatedwith AMLI-ETO, e.g., leukemia. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT3. In one example, the DAL1 tumorsuppressor protein has been shown to interact with PRMT3 and inhibitsits methyltransferase activity (Singh et al., Oncogene 2004 23,7761-7771). Epigenetic downregulation of DAL1 has been reported inseveral cancers (e.g., meningiomas and breast cancer), thus PRMT3 isexpected to display increased activity, and cancers that display DAL1silencing may, in some aspects, be good targets for PRMT3 inhibitors,e.g., those described herein. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT3, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT4, also known as CARM1. Forexample, PRMT4 levels have been shown to be elevated incastration-resistant prostate cancer (CRPC), as well as in aggressivebreast tumors (Hong et al., Cancer 2004 101, 83-89; Majumder et al.,Prostate 2006 66, 1292-1301). Thus, in some embodiments, inhibitors ofPRMT4, as described herein, are useful in treating cancers associatedwith PRMT4 overexpression. PRMT4 has also been shown to affectERα-dependent breast cancer cell differentiation and proliferation(Al-Dhaheri et al., Cancer Res. 2011 71, 2118-2128), thus in someaspects PRMT4 inhibitors, as described herein, are useful in treatingERα-dependent breast cancer by inhibiting cell differentiation andproliferation. In another example, PRMT4 has been shown to be recruitedto the promoter of E2F1 (which encodes a cell cycle regulator) as atranscriptional co-activator (Frietze et al., Cancer Res. 2008 68,301-306). Thus, PRMT4-mediated upregulation of E2F1 expression maycontribute to cancer progression and chemoresistance as increasedabundance of E2F1 triggers invasion and metastasis by activating growthreceptor signaling pathways, which in turn promote an antiapoptotictumor environment (Engelmann and Piitzer, Cancer Res 2012 72; 571).Accordingly, in some embodiments, the inhibition of PRMT4, e.g., bycompounds provided herein, is useful in treating cancers associated withE2F1 upregulation. Thus, without being bound by any particularmechanism, the inhibition of PRMT4, e.g., by compounds described herein,is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT6. For example, PRMT6 has beenreported to be overexpressed in a number of cancers, e.g., bladder andlung cancer (Yoshimatsu et al., Int. J. Cancer 2011 128, 562-573). Thus,in some embodiments, the inhibition of PRMT6, by compounds providedherein, is useful in treating cancers associated with PRMT6overexpression. In some aspects, PRMT6 is primarily thought to functionas a transcriptional repressor, although it has also been reported thatPRMT6 functions as a co-activator of nuclear receptors. For example, asa transcriptional repressor, PRMT6 suppresses the expression ofthrombospondin 1 (TSP1; also known as THBS1; a potent natural inhibitorof angiogenesis and endothelial cell migration) and p21 (a naturalinhibitor of cyclin dependent kinase), thereby contributing to cancerdevelopment and progression (Michaud-Levesque and Richard, J. Biol.Chem. 2009 284, 21338-21346; Kleinschmidt et al., PLoS ONE 2012 7,e41446). Accordingly, in some embodiments, the inhibition of PRMT6, bycompounds provided herein, is useful in treating cancer by preventingthe repression of THBs1 and/or p21. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT6, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT8. For example, deep-sequencingefforts of cancer genomes (e.g., COSMIC) have revealed that of all thePRMTs, PRMT8 is reported to be the most mutated. Of 106 sequencedgenomes, 15 carry mutations in the PRMT8 coding region, and nine ofthese result in an amino acid change (Forbes et al., Nucleic Acids Res.2011 39, D945-D950). Because of its high rate of mutation in cancer,PRMT8 is thought to contribute to the initiation or progression ofcancer. Thus, without being bound by any particular mechanism, theinhibition of PRMT8, e.g., by compounds described herein, is beneficialin the treatment of cancer.

In some embodiments, compounds described herein are useful for treatinga cancer including, but not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (e.g., “Waldenström's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphomalleukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemiallymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

In some embodiments, a compound provided herein is useful in treatingdiseases associated with increased levels of circulating asymmetricdimethylarginine (aDMA), e.g., cardiovascular disease, diabetes, kidneyfailure, renal disease, pulmonary disease, etc. Circulating aDMA isproduced by the proteolysis of asymmetrically dimethylated proteins.PRMTs which mediate aDMA methylation include, e.g., PRMT1, PRMT3, PRMT4,PRMT6, and PRMT8. aDMA levels are directly involved in various diseasesas aDMA is an endogenous competitive inhibitor of nitric oxide synthase(NOS), thereby reducing the production of nitric oxide (NO) (Vallance etal., J. Cardiovasc. Pharmacol. 1992 20(Suppl. 12):S60-2). NO functionsas a potent vasodilator in endothelial vessels, and as such inhibitingits production has major consequences on the cardiovascular system. Forexample, since PRMT1 is a major enzyme that generates aDMA, thedysregulation of its activity is likely to regulate cardiovasculardiseases (Boger et al., Ann. Med. 2006 38:126-36), and otherpathophysiological conditions such as diabetes mellitus (Sydow et al.,Vasc. Med. 2005 10(Suppl. 1):S35-43), kidney failure (Vallance et al.,Lancet 1992 339:572-5), and chronic pulmonary diseases (Zakrzewicz etal., BMC Pulm. Med. 2009 9:5). Additionally, it has been demonstratedthat the expression of PRMT1 and PRMT3 are increased in coronary heartdisease (Chen et al., Basic Res. Cardiol. 2006 101:346-53). In anotherexample, aDMA elevation is seen in patients with renal failure, due toimpaired clearance of this metabolite from the circulation (Jacobi etal., Am. J. Nephrol. 2008 28:224-37). Thus, circulating aDMA levels isobserved in many pathophysiological situations. Accordingly, withoutbeing bound by any particular mechanism, the inhibition of PRMTs, e.g.,by compounds described herein, results in the decrease of circulatingaDMA, which is beneficial in the treatment of diseases associated withincreased levels of circulating aDMA, e.g., cardiovascular disease,diabetes, kidney failure, renal disease, pulmonary disease, etc. Incertain embodiments, a compound described herein is useful for treatingor preventing vascular diseases.

In some embodiments, a compound provided herein is useful in treatingmetabolic disorders. For example, PRMT1 has been shown to enhance mRNAlevels of FoxO1 target genes in gluconeogenesis, which results inincreased hepatic glucose production, and knockdown of PRMT1 promotesinhibition of FoxO1 activity and thus inhibition of hepaticgluconeogenesis (Choi et al., Hepatology 2012 56:1546-56). Additionally,genetic haploinsufficiency of Prmt1 has been shown to reduce bloodglucose levels in mouse models. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, is beneficial in the treating of metabolic disorders,such as diabetes. In some embodiments, a provided compound is useful intreating type I diabetes. In some embodiments, a provided compound isuseful in treating type II diabetes.

In some embodiments, a compound provided herein is useful in treatingmuscular dystrophies. For example, PRMT1, as well as PRMT3 and PRMT6,methylate the nuclear poly(A)-binding protein (PABPN1) in a regionlocated near its C-terminus (Perreault et al., J. Biol. Chem. 2007282:7552-62). This domain is involved in the aggregation of the PABPN1protein, and abnormal aggregation of this protein is involved in thedisease oculopharyngeal muscular dystrophy (Davies et al., Int. J.Biochem. Cell. Biol. 2006 38:1457-62). Thus, without being bound by anyparticular mechanism, the inhibition of PRMTs, e.g., by compoundsdescribed herein, is beneficial in the treatment of musculardystrophies, e.g., oculopharyngeal muscular dystrophy, by decreasing theamount of methylation of PABPN1, thereby decreasing the amount of PABPN1aggregation.

CARM1 is also the most abundant PRMT expressed in skeletal muscle cells,and has been found to selectively control the pathways modulatingglycogen metabolism, and associated AMPK (AMP-activated protein kinase)and p38 MAPK (mitogen-activated protein kinase) expression. See, e.g.,Wang et al., Biochem (2012) 444:323-331. Thus, in some embodiments,inhibitors of CARM1, as described herein, are useful in treatingmetabolic disorders, e.g., for example skeletal muscle metabolicdisorders, e.g., glycogen and glucose metabolic disorders. Exemplaryskeletal muscle metabolic disorders include, but are not limited to,Acid Maltase Deficiency (Glycogenosis type 2; Pompe disease), Debrancherdeficiency (Glycogenosis type 3), Phosphorylase deficiency (McArdle's;GSD 5), X-linked syndrome (GSD9D), Autosomal recessive syndrome (GSD9B),Tarui's disease (Glycogen storage disease VII; GSD 7), PhosphoglycerateMutase deficiency (Glycogen storage disease X; GSDX; GSD 10), Lactatedehydrogenase A deficiency (GSD 11), Branching enzyme deficiency (GSD4), Aldolase A (muscle) deficiency, β-Enolase deficiency,Triosephosphate isomerase (TIM) deficiency, Lafora's disease(Progressive myoclonic epilepsy 2), Glycogen storage disease (Muscle,Type 0, Phosphoglucomutase 1 Deficiency (GSD 14)), and GlycogeninDeficiency (GSD 15).

In some embodiments, a compound provided herein is useful in treatingautoimmune disease. For example, several lines of evidence stronglysuggest that PRMT inhibitors may be valuable for the treatment ofautoimmune diseases, e.g., rheumatoid arthritis. PRMTs are known tomodify and regulate several critical immunomodulatory proteins. Forexample, post-translational modifications (e.g., arginine methylation),within T cell receptor signaling cascades allow T lymphocytes toinitiate a rapid and appropriate immune response to pathogens.Co-engagement of the CD28 costimulatory receptor with the T cellreceptor elevates PRMT activity and cellular protein argininemethylation, including methylation of the guanine nucleotide exchangefactor Vav1 (Blanchet et al., J. Exp. Med. 2005 202:371-377). PRMTinhibitors are thus expected to diminish methylation of the guanineexchange factor Vav1, resulting in diminished IL-2 production. Inagreement, siRNA directed against PRMT5 was shown to both inhibitNFAT-driven promoter activity and IL-2 secretion (Richard et al.,Biochem J. 2005 388:379-386). In another example, PRMT1 is known tocooperate with PRMT4 to enhance NFkB p65-driven transcription andfacilitate the transcription of p65 target genes like TNFα (Covic etal., Embo. J. 2005 24:85-96). Thus, in some embodiments, PRMT1 and/orPRMT4 inhibitors, e.g., those described herein, are useful in treatingautoimmune disease by decreasing the transcription of p65 target geneslike TNFα. These examples demonstrate an important role for argininemethylation in inflammation. Thus, without being bound by any particularmechanism, the inhibition of PRMTs, e.g., by compounds described herein,is beneficial in the treatment of autoimmune diseases.

In some embodiments, a compound provided herein is useful in treatingneurological disorders, such as amyotrophic lateral sclerosis (ALS). Forexample, a gene involved in ALS, TLS/FUS, often contains mutatedarginines in certain familial forms of this disease (Kwiatkowski et al.,Science 2009 323:1205-8). These mutants are retained in the cytoplasm,which is similar to reports documenting the role arginine methylationplays in nuclear-cytoplasmic shuffling (Shen et al., Genes Dev. 199812:679-91). This implicates PRMT, e.g., PRMT1, function in this disease,as it was demonstrated that TLS/FUS is methylated on at least 20arginine residues (Rappsilber et al., Anal. Chem. 2003 75:3107-14).Thus, in some embodiments, the inhibition of PRMTs, e.g., by compoundsprovided herein, are useful in treating ALS by decreasing the amount ofTLS/FUS arginine methylation.

In some embodiments, compounds of Formula (I) can be prepared usingmethods shown in Scheme 1. Scheme 1 shows a general synthesis route toindazole compounds of Formula (I), wherein R_(1′) and X′ are either thesame as R₁ and X or are substituents which serve as precursors forsynthetic conversion to R₁ and X wherein R₁, R_(x), R₃, X and Y are asdefined above. In the first step indazole-3-carboxylates of generalformula XI are reduced to alcohol intermediates of general formula XIIusing standard conditions, for example, using LiAlH₄ in THF undercooling or at room temperature. Intermediates XII can then be convertedto indazole-3-carboxaldehydes of general formula XIII by oxidation usingstandard conditions, for example, using MnO₂ in an aprotic solvent atroom or elevated temperature. Reaction of intermediates XIII withmono-Boc protected ethylenediamines of general formula XIV underreductive amination conditions (e.g. sodium cyanoborohydride andcatalytic acid such as acetic acid) in an appropriate solvent (e.g.methanol) allows the preparation of intermediates of general formula XV.In a subsequent optional step or steps, either R_(1′) and X′ can beconverted as required to the R₁ and X or substituents present in finalcompounds of Formula (I). For example, compounds with R₁ as an alkoxygroup can be synthesized by deprotection of the alkylation intermediatesof formula XV (e.g. deprecation of R_(1′)=OTBS gives the correspondingfree alcohol R_(1′)═OH), followed by alkylation reaction of the hydroxylgroup with a suitable alkylbromide or iodide in the presence of anappropriate base (e.g. potassium carbonate) in an organic solvent (e.g.tetrahydrofuran) at room or elevated temperature. In a finaldeprotection step the N-Boc protecting can be removed with an acid (e.g.HCl) in a suitable organic solvent (e.g. ethanol) to give compounds offormula I.

Indazole-3-carboxylates of general formula XI may be prepared from3-unsubstituted indazole starting materials of formula XXI as depictedin Scheme 2. Iodination of starting materials XXI, for example, usingN-iodosuccinimide in DMF at room temperature can be used to prepare3-iodoindazoles of general formula XII. Methoxycarbonylation ofintermediates XII under standard conditions using a carbonmonoxide in apressure reactor in the presence of a palladium catalyst (e.g.Pd(dppf)Cl2) in methanol with triethylamine at an elevated temperaturegives methyl indazole-3-carboxylates XIII.

In certain embodiments, X′ in the intermediates of formulas XI, XII,XIII and XV may bear a protecting group (e.g. THP) which is eitherremoved concomitantly in the final Boc deprotection step, or mayoptionally be converted in separate additional deprotection andalkylation steps to the final X group. For example, preparation ofN-substituted indazole isomer intermediate carboxaldehydes of generalstructure XIIIa and XIIIb is depicted in Scheme 3. Scheme 3 uses a THPprotected intermediate with R₁ as an alkoxy group. Deprotection of theTHP group then gives an intermediate which can be treated with a baseand an alkylating agent R₂I to give a mixture of indazole nitrogenalkylated isomers XIIIa and XIIIb, typically favoring isomer XIIIa.After chromatographic separation, the individual isomers XXXIa and XIIIbcan be converted as described in Scheme 1 to the corresponding N-alkylindazole isomer compounds captured in Formula (I).

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Synthetic Methods

General methods and experimental procedures for preparing andcharacterizing compounds of the present invention are set forth below.Wherever needed, reactions were heated using conventional hotplateapparatus or heating mantle or microwave irradiation equipment.Reactions were conducted with or without stirring, under atmospheric orelevated pressure in either open or closed vessels. Reaction progresswas monitored using conventional techniques such as TLC, HPLC, UPLC, orLCMS using instrumentation and methods described below. Reactions werequenched and crude compounds isolated using conventional methods asdescribed in the specific examples provided. Solvent removal was carriedout with or without heating, under atmospheric or reduced pressure,using either a rotary or centrifugal evaporator. Compound purificationwas carried out as needed using a variety of traditional methodsincluding, but not limited to, preparative chromatography under acidic,neutral, or basic conditions using either normal phase or reverse phaseHPLC or flash columns or Prep-TLC plates. Compound purity and massconfirmations were conducted using standard HPLC and/or UPLC and/or MSspectrometers and/or LCMS and/or GC equipment (e.g., including, but notlimited to the following instrumentation: Waters Alliance 2695 with 2996PDA detector connected with ZQ detector and ESI source; ShimadzuLDMS-2020; Waters Acquity H Class with PDA detector connected with SQdetector and ESI source; Agilent 1100 Series with PDA detector; WatersAlliance 2695 with 2998 PDA detector; AB SCIEX API 2000 with ESI source;Agilent 7890 GC). Exemplified compounds were dissolved in either MeOH orMeCN to a concentration of approximately 1 mg/mL and analyzed byinjection of 0.5-10 μL into an appropriate LCMS system using the methodsprovided in the following table:

MS Heat MS Flow Block Detector Mobile Mobile Rate Temp Voltage MethodColumn Phase A Phase B (mL/min) Gradient Profile (° C.) (kV) A Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 250 1.5 XR-ODS TFA TFA 2.0minutes, 2.2 μm 100% B for 1.1 3.0 × 50 mm minutes, 100% to 5% B in 0.2minutes, then stop B Gemini-NX Water/ ACN 1 5% to 100% B in 200 0.75 3μm C18 0.04% 2.0 minutes, 110A Ammonia 100% B for 1.1 minutes, 100% to5% B in 0.1 minutes, then stop C Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 250 0.85 XR-ODS FA FA 2.0 minutes, 1.6 μm 100% B for 1.1 2.0 ×50 mm minutes, 100% to 5% B in 0.1 minutes, then stop D Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 250 0.95 XR-ODS TFA TFA 2.0minutes, 2.2 μm 100% B for 1.1 3.0 × 50 mm minutes, 100% to 5% B in 0.1minutes, then stop E Waters Water/0.05% ACN/0.05% 0.9 5% to 100% B in250 1.5 Xselect C18 FA FA 2.0 minutes, 3.5 μm 100% B for 1.2 3.0 × 50 mmminutes, 100% to 5% B in 0.1 minutes, then stop F Shim-pack Water/0.05%ACN/0.05% 1 5% to 80% B in 200 0.95 XR-ODS TFA TFA 3.25 minutes, 2.2 μm80% B for 1.35 3.0 × 50 mm minutes, 80% to 5% B in 0.3 minutes, thenstop G Shim-pack Water/0.05% ACN/0.05% 1 5% to 70% B in 200 0.95 XR-ODSTFA TPF 2.50 minutes, 2.2 μm 70% B for 0.70 3.0 × 50 mm minutes, 70% to5% B in 0.1 minutes, then stop H Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 250 0.95 XR-ODS TFA TFA 2.20 minutes, 2.2 μm 100% B for 1.003.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop I Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 250 0.95 XR-ODS TFA TFA 1.20minutes, 2.2 μm 100% B for 1.00 3.0 × 50 mm minutes, 100% to 5% B in 0.1minutes, then stop J Shim-pack Water/0.05% ACN/0.05% 1 5% to 70% B in250 0.95 XR-ODS TFA TFA 3.20 minutes, 2.2 μm 70% B for 0.75 3.0 × 50 mmminutes, 70% to 5% B in 0.35 minutes, then stop K Shim-pack Water/0.05%ACN/0.05% 1 5% to 80% B in 250 1.5 XR-ODS TFA TFA 3.00 minutes, 2.2 μm80% B for 0.8 3.0 × 50 mm minutes, 80% to 5% B in 0.1 minutes, then stopL Shim-pack Water/0.05% ACN/0.05% 1 5% to 100% B in 250 1.5 XR-ODS TFATFA 3.00 minutes, 2.2 μm 100% B for 0.8 3.0 × 50 mm minutes, 100% to 5%B in 0.1 minutes, then stop M Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 250 1.5 XR-ODS TFA TFA 2.20 minutes, 2.2 μm 100% B for 1.003.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop N Shim-packWater/0.05% ACN/0.05% 1 5% to 80% B in 250 1.5 XR-ODS TFA TFA 2.20minutes, 2.2 μm 80% B for 1.0 3.0 × 50 mm minutes, 80% to 5% B in 0.1minutes, then stop O Zorbax Water/0.05% ACN/0.05% 1 5% to 70% B in 2501.5 Eclipse Plus TFA TFA 8.00 minutes, C18 70% B for 2.0 4.6 × 100 mmminutes, then stop P Shim-pack Water/0.05% ACN/0.05% 1 5% to 65% B in250 1.5 XR-ODS TFA TFA 3.00 minutes, 2.2 μm 65% B for 0.80 3.0 × 50 mmminutes, 100% to 5% B in 0.1 minutes, then stop Q Shim-pack Water/0.05%ACN/0.05% 1 5% to 60% B in 250 0.95 XR-ODS TFA TFA 2.50 minutes, 2.2 μm60% B for 0.7 3.0 × 50 mm minutes, 60% to 5% B in 0.1 minutes, then stopR Shim-pack Water/0.05% ACN/0.05% 1 5% to 50% B in 250 0.95 XR-ODS TFATFA 2.50 minutes, 2.2 μm 50% B for 0.7 3.0 × 50 mm minutes, 50% to 5% Bin 0.1 minutes, then stop S XBridge C18 Water/0.05% ACN/0.05% 1 5% to95% B in 250 0.9 3.5 μm TFA TFA 2.20 minutes, 3.0 × 50 mm 95% B for 1.00minutes, 95% to 5% B in 0.1 minutes, then stop T Shim-pack Water/0.05%ACN/0.05% 0.7 5% to 100% B in 250 0.85 XR-ODS FA FA 2.0 minutes, 1.6 μm100% B for 1.1 2.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, thenstop U Shim-pack Water/0.05% ACN/0.05% 1 5% to 40% B in 250 0.95 XR-ODSTFA TFA 2.50 minutes, 2.2 μm 40% B for 0.7 3.0 × 50 mm minutes, 40% to5% B in 0.1 minutes, then stop V Shim-pack Water/0.05% ACN/0.05% 1 5% to60% B in 200 1.05 XR-ODS TFA TFA 4.20 minutes, 2.2 μm 60% B for 1.0 3.0× 50 mm minutes, 60% to 5% B in 0.1 minutes, then stop W Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 200 0.95 XR-ODS TFA TFA 2.20minutes, 2.2 μm 100% B for 1.00 3.0 × 50 mm minutes, 100% to 5% B in 0.1minutes, then stop X Shim-pack Water/0.05% ACN/0.05% 0.7 5% to 100% B in200 0.85 XR-ODS FA FA 2.0 minutes, 1.6 μm 100% B for 1.1 2.0 × 50 mmminutes, 100% to 5% B in 0.1 minutes, then stop Y Ecliplis PlusWater/0.05% ACN 1 5% to 100% B in 250 1 C18 3.5 μm TFA 2.0 minutes, 4.6× 50 mm 100% B for 1.0 minutes, 100% to 5% B in 0.1 minutes, then stop ZEcliplis Plus Water/10 mM ACN/5% 1 5% to 100% B in 250 1.1 C18 3.5 μmammonium water 2.0 minutes, 4.6 × 50 mm carbonate 100% B for 1.0minutes, 100% to 5% B in 0.1 minutes, then stop A1 Shim-pack Water/0.05%ACN 1 5% to 100% B in 250 1 XR-ODS TFA 2.0 minutes, 2.2 μm 100% B for1.0 3.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop A2Ecliplis Plus Water/10 mM ACN 1 5% to 100% B in 250 0.95 C18 3.5 μmammonium 2.0 minutes, 4.6 × 50 mm acetate 100% B for 1.4 minutes, 100%to 5% B in 0.1 minutes, then stop A3 Acquity BEH Water/5 mM ACN/0.1%0.55 5% B at 0.01 min C18 1.7 μm ammonium FA up to 0.4 min, 2.1 × 50 mmacetate/ 35% B at 0.8 min, 0.1% FA 55% B at 1.2 min, 100% B in 1.3minutes, at 2.5 min up to 3.30 min, 5% B at 3.31 min up to 4.0 min, thenstop

Compound structure confirmations were carried out using standard 300 or400 MHz NMR spectrometers with NOe's conducted whenever necessary.

The following abbreviations are used herein:

Abbreviation Meaning ACN acetonitrile atm. atmosphere DCMdichloromethane DHP dihydropyran DIBAL diisobutyl aluminum hydride DIEAdiisopropyl ethylamine DMF dimethyl formamide DMF-DMA dimethyl formamidedimethyl acetal DMSO dimethyl sulfoxide dppf1,1′-bis(diphenylphosphino)ferrocene EA ethyl acetate ESI electrosprayionization EtOH ethanol FA formic acid GC gas chromatography h hour Hexhexanes HMDS hexamethyl disilazide HPLC high performance liquidchromatography IPA isopropanol LCMS liquid chromatography/massspectrometry MeOH methanol min minutes NBS N-bromo succinimide NCSN-chloro succinimide NIS N-iodo succinimide NMR nuclear magneticresonance NOe nuclear Overhauser effect Prep. preparative PTSApara-toluene sulfonic acid Rf retardation factor rt room temperature RTretention time sat. saturated SGC silica gel chromatography TBAFtetrabutyl ammonium fluoride TEA triethylamine TFA trifluoroacetic acidTHF tetrahydrofuran TLC thin layer chromatography UPLC ultra performanceliquid chromatography

Compound 2N1-((5-iso-butoxy-1H-indazol-3-yl)methyl)-N1-methylethane-1,2-diamine

Step 1 5-(tert-butyldimethylsilyloxy)-1H-indazole

A solution of 1H-indazol-5-ol (25 g, 186.38 mmol, 1.00 equiv), imidazole(31.7 g, 466.18 mmol, 2.50 equiv) and tert-butyl(chloro)dimethylsilane(33.8 g, 224.25 mmol, 1.20 equiv) in N,N-dimethylformamide (100 mL) wasstirred at room temperature for 2 h. The reaction was quenched by theaddition of water/ice (200 mL). The precipitate was collected byfiltration and dried in a vacuum oven to give 45 g (97%) of5-(tert-butyldimethylsilyloxy)-1H-indazole as a brown solid. 1H NMR (300MHz, CDCl₃): δ 8.00 (s, 1H), 7.40 (d, J=9.0 Hz, 1H), 7.15 (d, J=9.0 Hz,1H), 7.02 (dd, J=9.0 Hz, 2.4 Hz, 1H), 1.03 (s, 9H), 0.24 (s, 6H) ppm.LCMS (method D, ESI): RT=1.66 min, m/z=249.0 [M+H]⁺.

Step 2 5-(tert-butyldimethylsilyloxy)-3-iodo-1H-indazole

To a solution of 5-[(tert-butyldimethylsilyl)oxy]-1H-indazole (13 g,52.34 mmol, 1.00 equiv) in N,N-dimethylformamide (550 mL) maintainedunder nitrogen was added NIS (28.6 g, 104.76 mmol, 2.00 equiv) inportions. The reaction mixture was stirred at room temperature overnightand then concentrated under vacuum. The residue was purified on a silicagel column eluted with 0-10% of ethyl acetate in petroleum ether to give19.2 g (98%) of 5-(tert-butyldimethylsilyloxy)-3-iodo-1H-indazole as anoff-white solid. 1H NMR (300 MHz, CDCl₃): δ 7.50-7.45 (m, 1H), 7.09 (dd,J=9.0 Hz, 2.1 Hz, 1H), 6.98-6.70 (m, 1H), 1.10 (s, 9H), 0.24 (s, 6H)ppm. LCMS (method A, ESI): RT=1.80 min, m/z=374.9 [M+H]⁺.

Step 3 (R/S)5-(tert-butyldimethylsilyloxy)-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

A solution of (R/S) 5-(tert-butyldimethylsilyloxy)-3-iodo-1H-indazole(9.6 g, 25.65 mmol, 1.00 equiv), 3,4-dihydro-2H-pyran (6.47 g, 77.02mmol, 3.00 equiv) and p-toluenesulfonic acid (440 mg, 2.56 mmol, 0.10equiv) in THF (100 mL) was stirred at room temperature for 3 h. Thereaction mixture was diluted with 100 mL of H₂O and then extracted with3×200 mL of dichloromethane. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 0-10% of ethyl acetate inpetroleum ether to give 11 g (94%) of (R/S)5-(tert-butyldimethylsilyloxy)-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazoleas a light brown oil. LCMS (method D, ESI): RT=1.45 min, m/z=459.0[M+H]⁺.

Step 4 (R/S) methyl5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carboxylate

A mixture of (R/S)5-(tert-butyldimethylsilyloxy)-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(5 g, 10.91 mmol, 1.00 equiv), Pd(dppf)Cl2 (800 mg, 1.09 mmol, 0.10equiv) and triethylamine (3.3 g, 32.61 mmol, 2.99 equiv) in methanol (50mL) was stirred under 10 atm. of carbon monoxide in a 100-mL pressurereactor at 60° C. for 5 h. The reaction was cooled to room temperaturethen concentrated under vacuum. The residue was diluted with 20 mL ofH₂O and the resulting mixture was extracted with 3×50 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 0-7% of ethyl acetate in petroleumether to yield 2.6 g (61%) of (R/S) methyl5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carboxylateas a colorless oil. 1H NMR (300 MHz, CDCl₃): δ 7.62-7.57 (m, 2H), 7.04(dd, J=9.0 Hz, 2.4 Hz, 1H), 5.82-5.78 (m, 1H), 3.85-3.70 (m, 2H),3.68-3.47 (m, 1H), 2.63-2.45 (m, 1H), 2.10 (s, 3H), 1.08-1.05 (m, 4H),1.05 (s, 9H), 0.25 (s, 6H) ppm. LCMS (method E, ESI): RT=1.90 min,m/z=391.1 [M+H]⁺.

Step 5 (R/S)(5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methanol

To a stirred mixture of LiAH₄ (390 mg, 11.50 mmol, 1.00 equiv) inanhydrous THF (100 mL) maintained under nitrogen at −70° C. was addeddropwise a solution of (R/S) methyl5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carboxylate(1.33 g, 3.41 mmol, 1.00 equiv) in THF. The reaction was stirred at −40°C. for 1 h and then quenched by the addition of 20 mL of saturated NH₄Clsolution. The resulting mixture was concentrated under vacuum to removethe excess THF. The mixture was extracted with 3×50 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 0-20% of ethyl acetate in petroleumether to afford 1 g (81%) of (R/S)(5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methanolas a white solid. 1H NMR (300 MHz, CDCl₃): δ 7.22 (d, J=9.0 Hz, 1H),6.95 (d, J=2.1 Hz, 1H), 6.80 (dd, J=8.7 Hz, 2.1 Hz, 1H), 5.44-5.40 (m,1H), 4.80 (s, 2H), 3.80-3.71 (m, 2H), 2.01-1.75 (m, 2H), 1.69-1.25 (m,4H), 0.80 (s, 9H), 0.10 (s, 6H) ppm. LCMS (method A, ESI): RT=1.71 min,m/z=363.0 [M+H]⁺.

Step 6 (R/S)5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde

A mixture of (R/S)(5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methanol(1 g, 2.76 mmol, 1.00 equiv) and MnO₂ (2.4 g, 27.61 mmol, 10.01 equiv)in dichloromethane (10 mL) was stirred at room temperature for 5 h. Thesolid material was removed by filtration. The filtrate was concentratedunder vacuum to give 0.8 g (80%) of5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehydeas a colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 10.0 (s, 1H), 7.44 (s,1H), 7.29 (d, J=8.8 Hz, 1H), 6.81 (d, J=1.2 Hz, 1H), 5.57-5.54 (m, 1H),3.85-3.70 (m, 2H), 3.42-3.20 (m, 2H), 1.70-1.35 (m, 4H), 0.80 (s, 9H),0.10 (s, 6H) ppm. LCMS (method A, ESI): RT=1.88 min, m/z=360.9 [M+H]⁺.

Step 7 (R/S)5-hydroxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde

A solution of (R/S)5-(tert-butyldimethylsilyloxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde(150 mg, 0.42 mmol, 1.00 equiv) and TBAF (800 mg, 800.00 mmol, 1922.79equiv) in THF (5 mL) was stirred at room temperature for 2 h. Thereaction mixture was diluted with 10 mL of H₂O and then extracted with3×20 mL of dichloromethane. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 10-20% of ethyl acetate inpetroleum ether to give 0.1 g (98%) of (R/S)5-hydroxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde as anoff-white solid. LCMS (method A, ESI): RT=1.33 min, m/z=246.0 [M+H]⁺.

Step 85-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde

A mixture of (R/S)5-hydroxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde (200mg, 0.81 mmol, 1.00 equiv), 1-iodo-2-methylpropane (300 mg, 1.63 mmol,2.01 equiv) and Cs₂CO₃ (270 mg, 0.83 mmol, 1.02 equiv) inN,N-dimethylformamide (10 mL) was stirred under nitrogen at 70° C. for 5h. The resulting solution was cooled to room temperature and dilutedwith 10 mL of H₂O. The mixture was extracted with 3×20 mL of ethylacetate. The combined organic layers were washed with 3×20 mL of brine,dried over anhydrous sodium sulfate and concentrated under vacuum. Theresidue was purified on a silica gel column eluted with 0-15% of ethylacetate in petroleum ether to give 0.165 g (67%) of (R/S)5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde asa white solid. ¹H-NMR (300 MHz, DMSO-d6): δ 10.16 (s, 1H), 7.83 (d,J=9.3 Hz, 1H), 7.51 (d, J=2.1 Hz, 1H), 7.22 (dd, J=9.3 Hz, 2.4 Hz, 1H),6.04-6.01 (m, 1H), 4.07-4.00 (m, 1H), 3.92-3.75 (m, 4H), 2.12-2.00 (m,2H), 1.63-1.62 (m, 2H), 1.24-1.16 (m, 2H), 1.04 (d, J=6.6 Hz, 6H) ppm.LCMS (method A, ESI): RT=1.71 min, m/z=218.9 [M+H-THP]+.

Step 9 (R/S) tert-butyl2-(((5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of (R/S)5-(2-methylpropoxy)-1-(oxan-2-yl)-1H-indazole-3-carbaldehyde (200 mg,0.66 mmol, 1.00 equiv) and tert-butyl N-[2-(methylamino)ethyl]carbamate(140 mg, 0.80 mmol, 1.21 equiv) in 1,2-dichloroethane (10 mL) was addedNaBH(AcO)₃ (420 mg, 1.98 mmol, 3.00 equiv). The resulting solution wasstirred at room temperature for 3 h and then diluted with 10 mL of H₂O.The mixture was extracted with 3×20 mL of dichloromethane. The combinedorganic layers was dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was purified on a silica gel column elutedwith 10-50% of ethyl acetate in petroleum ether to give 0.15 g (49%) of(R/S) tert-butyl2-(((5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)carbamateas a colorless oil. ¹H-NMR (300 MHz, CDCl₃): δ 7.55-7.40 (m, 1H),7.21-7.00 (m, 2H), 5.75-5.50 (m, 1H), 5.30 (s, 2H), 4.20-3.85 (m, 2H),3.85-3.55 (m, 2H), 3.50-3.10 (m, 2H), 2.05 (s, 3H), 1.97-1.60 (m, 4H),1.44 (s, 9H), 1.38-1.19 (m, 5H), 1.05 (d, J=6.6 Hz, 6H) ppm. LCMS(method A, ESI): RT=3.37 min, m/z=461.1 [M+H]⁺.

Step 10 Compound 2N¹-((5-iso-butoxy-1H-indazol-3-yl)methyl)-N¹-methylethane-1,2-diamine

A solution of (R/S) tert-butyl2-(((5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)carbamate(150 mg, 0.33 mmol, 1.00 equiv) in 3N hydrochloric acid (10 mL) wasstirred at 60° C. for 3 h. The resulting mixture was concentrated undervacuum and the crude product was purified by Prep-HPLC with thefollowing conditions (Waters-1): Column, XBridge Shield RP 18, 5μm,19×150 mm; mobile phase, phase A: water with 0.2% TFA; phase B: CH₃CN(10% CH₃CN up to 43% in 10 min, up to 100% in 13 min; Detector, UV 254nm to give 67.9 mg (41%) ofN¹-((5-iso-butoxy-1H-indazol-3-yl)methyl)-N¹-methylethane-1,2-diaminetrifluoroacetate as a light brown solid. ¹H-NMR (300 MHz, D₂O): δ 7.57(d, J=9.0 Hz, 1H), 7.24-7.17 (m, 2H), 4.74-4.71 (m, 2H), 3.84 (d, J=6.6Hz, 2H), 3.58-3.42 (m, 4H), 2.92 (s, 3H), 2.07-1.98 (m, 1H), 0.96 (d,J=6.6 Hz, 6H) ppm. LCMS (method X, ESI): RT=0.82 min, m/z=277.1 [M+H]+.

Compound 3N¹-((5-iso-butoxy-1-methyl-1H-indazol-3-yl)methyl)-N¹,N²-dimethylethane-1,2-diamine

Step 1 (R/S)5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde

A mixture of (R/S) 5-hydroxy-1-(oxan-2-yl)-1H-indazole-3-carbaldehyde(590 mg, 2.40 mmol, 1.00 equiv), 1-iodo-2-methylpropane (882.6 mg, 4.80mmol, 2.00 equiv) and cesium carbonate (782 mg, 2.39 mmol, 1.00 equiv)and N,N-dimethylformamide (15 mL) was stirred under nitrogen overnightat 60° C. The reaction was cooled to room temperature then quenched bythe addition of 50 mL of water/ice. The resulting mixture was extractedwith 3×50 mL of ethyl acetate. The combined organic layers was washedwith 3×50 mL of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 15-35% of ethyl acetate in petroleum ether to give250 mg (35%) of (R/S)5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde asa yellow solid. 1H NMR (300 MHz, CDCL₃): δ 10.25 (s, 1H), 7.65 (d, J=2.1Hz, 1H), 7.59 (d, J=9.0 Hz, 1H), 7.16 (dd, J=9.0 Hz, 2.1 Hz, 1H), 5.83(d, J=6.9 Hz, 1H), 4.05 (m, 1H), 3.83 (d, J=6.6 Hz, 2H), 3.80 (m, 1H),2.60 (m, 1H), 2.15 (m, 3H), 1.80 (m, 3H), 1.08 (d, J=6.9 Hz, 6H) ppm.LCMS (method D, ESI): RT=1.77 min, m/z=303.1 [M+H]⁺.

Step 2 5-iso-butoxy-1H-indazole-3-carbaldehyde

A solution of5-iso-butoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde(250 mg, 0.83 mmol, 1.00 equiv) in 12N hydrochloric acid (3 mL), ethanol(3 mL) and 1,4-dioxane (6 mL) was stirred overnight at room temperature.The reaction mixture was concentrated under vacuum and the pH value ofthe solution was adjusted to 8 with saturated sodium bicarbonatesolution. The resulting mixture was extracted with 3×50 mL ofdichloromethane. The organic layers were combined, dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 60-100% of ethyl acetate in petroleumether to yield 110 mg (61%) of 5-iso-butoxy-1H-indazole-3-carbaldehydeas a yellow solid. 1H NMR (300 MHz, DMSO-d₆): δ 10.16 (s, 1H), 7.61 (d,J=9.0 Hz, 1H), 7.48 (d, J=2.1 Hz, 1H), 7.14 (dd, J=9.0 Hz, 2.1 Hz, 1H),3.80 (d, J=6.6 Hz, 1H), 2.10-2.05 (m, 1H), 1.01 (d, J=6.6 Hz, 6H) ppm.LCMS (method D, ESI): RT=1.50 min, m/z=219.1 [M+H]+.

Step 3 5-iso-butoxy-1-methyl-1H-indazole-3-carbaldehyde

A mixture of 5-iso-butoxy-1H-indazole-3-carbaldehyde (110 mg, 0.50 mmol,1.00 equiv), potassium carbonate (209 mg, 1.50 mmol, 2.98 equiv) andmethyl iodide (107.5 mg, 0.76 mmol, 1.50 equiv) in acetonitrile (4 mL)was stirred at room temperature overnight. The resulting mixture wasconcentrated under vacuum and the residue was filtered through a silicagel column eluted with 2-5% of ethyl acetate in petroleum ether. Thecrude product (70 mg) was purified by Pre-HPLC with the followingconditions (1#-Pre-HPLC-005(Waters)): Column, XBridge Shield RP18 OBDColumn, 5 μm, 19×150 mm; mobile phase, water with 10 mmol NH₄HCO₃ andCH₃CN (18% CH₃CN up to 58% in 10 min, up to 95% in 1 min, down to 18% in2 min); Detector, UV 254/220 nm to give 35 mg (30%) of5-iso-butoxy-1-methyl-1H-indazole-3-carbaldehyde as a white solid. 1HNMR (400 MHz, CDCl₃): δ 10.1 (s, 1H), 7.55 (d, J=2.0 Hz, 1H), 7.27 (d,J=8.8 Hz, 1H), 7.07 (dd, J=8.8 Hz, 2.0 Hz, 1H), 4.08 (s, 3H), 3.74 (d,J=6.8 Hz, 2H), 2.09-2.02 (m, 1H), 0.98 (d, J=6.8 Hz, 6H) ppm. LCMS(method D, ESI): RT=2.23 min, m/z=233.1 [M+H]⁺ and 15 mg (13%) of5-iso-butoxy-2-methyl-2H-indazole-3-carbaldehyde as a white solid. 1HNMR (400 MHz, CDCl₃): □ 10.12 (s, 1H), 7.62 (d, J=9.2 Hz, 1H), 7.17 (d,J=2.4 Hz, 1H), 7.07 (dd, J=9.2 Hz, 2.4 Hz, 1H), 4.38 (s, 3H), 3.73 (d,J=6.4 Hz, 2H), 2.10-2.05 (m, 1H), 0.99 (d, J=6.8 Hz, 6H) ppm. LCMS(method D, ESI): RT=2.24 min, m/z=233.1[M+H]⁺.

Step 4 tert-butyl2-(((5-iso-butoxy-1-methyl-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

To a solution of 5-iso-butoxy-1-methyl-1H-indazole-3-carbaldehyde (35mg, 0.15 mmol, 1.00 equiv) and tert-butylN-methyl-N-[2-(methylamino)ethyl]carbamate (34 mg, 0.18 mmol, 1.20equiv) in 1,2-dichloroethane (3 mL) was added NaBH(OAc)₃ (96 mg, 0.45mmol, 3.01 equiv). The resulting solution was stirred at roomtemperature for 8 h then quenched with 20 mL of water/ice. The resultingmixture was extracted with 3×20 mL of dichloromethane. The organiclayers were combined, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-100% of ethyl acetate to give 48 mg (79%) oftert-butyl2-(((5-iso-butoxy-1-methyl-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamateas a colorless oil. LCMS (method C, ESI): RT=0.86 min, m/z=405.3 [M+H]⁺.

Step 5 Compound 3N¹-((5-iso-butoxy-1-methyl-1H-indazol-3-yl)methyl)-N¹,N²-dimethylethane-1,2-diamine

A solution of tert-butyl2-(((5-iso-butoxy-1-methyl-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(48 mg, 0.12 mmol, 1.00 equiv) in 3N hydrochloric acid (10 mL) wasstirred at 60° C. for 2 h. The resulting solution was washed with 3×50mL of dichloromethane. The aqueous layer was concentrated under vacuumand the crude product was purified by Prep-HPLC with the followingconditions (2#-Waters 2767-2(HPLC-08)): Column, XBridge Shield RP 18, 5μm, 19×150 mm; mobile phase, water with 50 mmol CF₃COOH and CH₃CN (10.0%CH₃CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1min, down to 10.0% in 1 min); Detector, UV 254 nm to give 31.3 mg (50%)ofN¹-((5-iso-butoxy-1-methyl-1H-indazol-3-yl)methyl)-N¹,N²-dimethylethane-1,2-diaminetrifluoroacetate as a colorless oil. ¹H NMR (300 MHz, D₂O): δ 7.53 (d,J=10.5 Hz, 1H), 7.23-7.21 (m, 2H), 4.68-4.65 (m, 2H), 4.02 (s, 3H), 3.83(d, J=6.9 Hz, 2H), 3.63-3.44 (m, 4H), 2.89 (s, 3H), 2.72 (s, 3H),2.10-1.93 (m, 1H), 0.94 (d, J=6.6 Hz, 6H) ppm. LCMS (method W, ESI):RT=1.31 min, m/z=305.1 [M+H]⁺.

Compound 5N¹-((5-(2-methoxyethoxy)-1H-indazol-3-yl)methyl)-N¹,N²-dimethylethane-1,2-diamine

Step 1 (R/S)5-(2-methoxyethoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde

A solution of 5-hydroxy-1-(oxan-2-yl)-1H-indazole-3-carbaldehyde (120mg, 0.49 mmol, 1.00 equiv), Cs₂CO₃ (180 mg, 0.55 mmol, 2.00 equiv) and1-bromo-2-methoxyethane (500 mg, 3.60 mmol, 3.00 equiv) inN,N-dimethylformamide (2 mL) was stirred at room temperature for 10 h.The reaction was diluted with 50 mL of ethyl acetate and then washedwith 4×10 mL of brine. The organic layer was dried over anhydrous sodiumsulfate then concentrated under vacuum to give 134 mg (90%) of (R/S)5-(2-methoxyethoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehydeas a yellow oil. LCMS (method D, ESI): RT=1.48 min, m/z=305.1 [M+H]+.

Step 2 (R/S) tert-butyl2-(((5-(2-methoxyethoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

To a solution of (R/S)5-(2-methoxyethoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde(130 mg, 0.43 mmol, 1.00 equiv) and tert-butylN-methyl-N-[2-(methylamino)ethyl]carbamate (180 mg, 0.96 mmol, 2.50equiv) in 1,2-dichloroethane (3 mL) was added NaBH(OAc)₃ (260 mg, 1.23mmol, 3.00 equiv). The resulting solution was stirred at roomtemperature for 2 h. The pH value of the solution was adjusted to 10with 5% potassium carbonate solution. The resulting solution wasextracted with 4×10 mL of dichloromethane. The combined organic layerswas concentrated under vacuum to give 156 mg (77%) of (R/S) tert-butyl2-(((5-(2-methoxyethoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamateas a yellow oil. LCMS (method A, ESI): RT=1.32 min, m/z=477.2 [M+H]⁺.

Step 3 Compound 5N¹-((5-(2-methoxyethoxy)-1H-indazol-3-yl)methyl)-N¹,N²-dimethylethane-1,2-diamine

A solution of (R/S) tert-butylN-[2-([[5-(2-methoxyethoxy)-1-(oxan-2-yl)-1H-indazol-3-yl]methyl](methyl)amino)ethyl]-N-methylcarbamate(156 mg, 0.33 mmol, 1.00 equiv) in trifluoroacetic acid (3 mL) anddichloromethane (2 mL) was stirred at room temperature for 4 h. Thereaction mixture was concentrated under vacuum and the crude product waspurified by Prep-HPLC with the following conditions: Column, XBridgeShield RP 18, 5 μm, 19×150 mm; mobile phase, water with 50 mmol CF₃COOHand CH₃CN (10.0% CH₃CN up to 28.0% in 2 min, up to 46.0% in 10 min, upto 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to give122.3 mg (70%) ofN¹-((5-(2-methoxyethoxy)-1H-indazol-3-yl)methyl)-N¹,N²⁻dimethylethane-1,2-diaminetrifluoroacetate as a colorless oil. ¹H-NMR (300 MHz, D₂O): δ 7.54 (d,J=9.0 Hz, 1H), 7.22 (d, J=1.8 Hz, 1H), 7.16 (dd, J=9.0 Hz, 2.1 Hz, 1H),4.78-4.72 (m, 2H), 4.20-4.13 (m, 2H), 3.80-3.74 (m, 2H), 3.60-3.42 (m,4H), 3.36 (s, 3H), 2.89 (s, 3H), 2.70 (s, 3H) ppm. LCMS (method M, ESI):RT=1.00 min, m/z=293.0 [M+H]⁺.

Compound 10N¹,N²-dimethyl-N¹-((5-phenoxy-1H-indazol-3-yl)methyl)ethane-1,2-diamine

Step 1 (R/S)5-(4-nitrophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde

A mixture of 1-fluoro-4-nitrobenzene (258 mg, 1.83 mmol, 1.00 equiv),(R/S) 5-hydroxy-1-(oxan-2-yl)-1H-indazole-3-carbaldehyde (300 mg, 1.22mmol, 0.67 equiv) and Cs₂CO₃ (1.2 g, 3.68 mmol, 2.01 equiv) in1,4-dioxane (25 mL) was stirred at 100° C. for 4 h. The reaction mixturewas cooled to room temperature and then diluted with 50 mL of H₂O. Theresulting mixture was extracted with 3×200 mL of ethyl acetate. Thecombined organic layers was dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-13% of ethyl acetate in petroleum ether to give 350mg (52%) of (R/S)5-(4-nitrophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehydeas a light yellow solid. ¹H-NMR (300 MHz, CDCl₃): δ 10.3 (s, 1H),8.23-8.18 (m, 2H), 8.01 (s, 1H), 7.78-7.75 (m, 1H), 7.27-7.23 (m, 1H),7.04-7.00 (m, 1H), 5.89-5.85 (m, 1H), 4.06-4.01 (m, 1H), 3.85-3.77 (m,1H), 2.60-2.52 (m, 1H), 2.21-2.17 (m, 2H), 1.86-1.74 (m, 3H) ppm.

Step 2 (R/S) tert-butylmethyl(2-(methyl((5-(4-nitrophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)amino)ethyl)carbamate

To a solution of (R/S)5-(4-nitrophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carbaldehyde(300 mg, 0.82 mmol, 1.00 equiv) and tert-butylN-methyl-N-[2-(methylamino)ethyl]carbamate (170 mg, 0.90 mmol, 1.11equiv) in 1,2-dichloroethane (40 mL) was added NaBH(OAc)₃ (520 mg). Thereaction was stirred at room temperature overnight and then quenchedwith 50 mL of water. The resulting mixture was extracted with 3×200 mLof ethyl acetate. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 0-10% of ethyl acetate in petroleumether to give 420 mg (95%) of (R/S) tert-butylmethyl(2-(methyl((5-(4-nitrophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)amino)ethyl)carbamateas a colorless oil. ¹H-NMR (400 MHz, CDCl₃): δ 8.18 (m, 2H), 7.71-7.55(m, 2H), 7.20-7.09 (m, 1H), 7.05-6.93 (m, 2H), 5.70-5.68 (m, 1H),4.11-4.09 (m, 1H), 4.06-3.91 (m, 1H), 3.79-3.73 (m, 1H), 3.50-3.33 (m,1H), 2.79 (s, 3H), 2.57-2.52 (m, 2H), 2.49-2.17 (m, 2H), 2.17-2.05 (m,1H), 1.80-1.68 (m, 4H), 1.42 (s, 9H) ppm.

Step 3 (R/S) tert-butyl2-(((5-(4-aminophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

A mixture of (R/S) tert-butylmethyl(2-(methyl((5-(4-nitrophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)amino)ethyl)carbamate(550 mg, 1.02 mmol, 1.00 equiv) and Raney/Ni (500 mg) in methanol (30mL) was stirred under hydrogen at room temperature for 30 min. Thecatalyst was removed by filtration. The filtrate was concentrated undervacuum to give 400 mg (77%) of (R/S) tert-butyl2-(((5-(4-aminophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl(methyl)carbamateas a colorless oil. ¹H-NMR (300 MHz, CDCl₃): δ 7.52-7.49 (m, 2H),7.33-7.26 (m, 2H), 7.18-7.12 (m, 1H), 6.90-6.80 (m, 2H), 6.71-6.61 (m,2H), 5.70-5.68 (m, 1H), 4.11-4.09 (m, 1H), 4.07-3.91 (m, 1H), 3.79-3.73(m, 1H), 3.71-3.33 (m, 1H), 2.79 (s, 3H), 2.57-2.52 (m, 2H), 2.49-2.17(m, 2H), 2.17-2.05 (m, 1H), 1.80-1.68 (m, 4H), 1.42 (s, 9H) ppm.

Step 4 (R/S) tert-butylmethyl(2-(methyl((5-phenoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)amino)ethyl)carbamate

To a stirred solution of (R/S) tert-butyl2-(((5-(4-aminophenoxy)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(400 mg, 0.78 mmol, 1.00 equiv) in hypophosphorous acid (10 mL) at 0° C.was added dropwise a solution of sodium nitrite (135 mg, 1.96 mmol, 2.49equiv) in water (5 mL). The reaction was stirred at 0° C. for 30 minthen the pH value of the solution was adjusted to 8 with saturatedsodium carbonate solution. The resulting mixture was extracted with3×200 mL of ethyl acetate. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 0-12.5% of ethyl acetate inpetroleum ether to give 300 mg (77%) of (R/S) tert-butylmethyl(2-(methyl((5-phenoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)amino)ethyl)carbamateas a colorless oil. LCMS (method A, ESI): RT=1.40 min, m/z=495.2 [M+H]⁺.

Step 5 Compound 10N¹,N²-dimethyl-N¹-((5-phenoxy-1H-indazol-3-yl)methyl)ethane-1,2-diamine

A solution of (R/S) tert-butylmethyl(2-(methyl((5-phenoxy-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)amino)ethyl)carbamate(250 mg, 0.51 mmol, 1.00 equiv) in 3N hydrochloric acid (20 mL) wasstirred at room temperature overnight. The reaction was concentratedunder vacuum and the residue was purified by Pre-HPLC with the followingconditions (1#-Pre-HPLC-005 (Waters)): Column, SunFire Prep C18 OBDColumn, 5μm, 19×150 mm; mobile phase, phase A: water with 0.05% TFA.Phase B: MeCN (5% CH₃CN up to 17% in 10 min, down to 0% in 0 min);Detector, UV 254/220 nm to afford 62.3 mg (23%) ofN¹,N²-dimethyl-N¹-((5-phenoxy-1H-indazol-3-yl)methyl)ethane-1,2-diaminetrifluoroacetate as a white oil. ¹H-NMR (300 MHz, D₂O): δ 7.68 (d, J=9.0Hz, 1H), 7.42-7.35 (m, 3H), 7.32-7.25 (m, 1H), 7.15 (t, J=7.2 Hz, 1H),7.02 (d, J=7.8 Hz, 2H), 4.70 (s, 2H), 3.60-3.43 (m, 4H), 2.88 (s, 3H),2.71 (s, 3H) ppm. LCMS (method M, ESI): RT=1.25 min, m/z=311.1 [M+H]⁺.

Synthesis of Intermediates Intermediate I tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

Step 1 tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (3.2 g,10.45 mmol, 1.00 equiv), tert-butyl N-[2-(methylamino)ethyl]carbamate(2.2 g, 12.63 mmol, 1.21 equiv) and NaBH(OAc)₃ (6.65 g, 31.38 mmol, 3.00equiv) in dichloroethane (30 mL) was stirred for 2 h at roomtemperature. The reaction was quenched with 50 mL of saturated aqueoussodium bicarbonate solution. The resulting mixture was extracted with3×200 mL of dichloromethane. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 30-100% ethyl acetate inpetroleum ether to give 4.05 g (83%) of tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamateas a light yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.48 (s, 1H),5.35-5.30 (m, 1H), 4.13-4.03 (m, 1H), 3.71-3.63 (m, 1H), 3.36 (s, 2H),3.26-3.25 (m, 2H), 2.52-2.49 (m, 2H), 2.21 (s, 3H), 2.09-2.01 (m, 3H),1.68-1.58 (m, 3H), 1.44 (s, 9H) ppm. LCMS (method C, ESI): RT=0.58 min,m/z=465.0 [M+H]⁺.

Intermediate II tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

Step 1 ethyl 3-iodo-1H-pyrazole-4-carboxylate

To a stirred solution of ethyl 3-amino-1H-pyrazole-4-carboxylate (10 g,64.45 mmol, 1.00 equiv) in 50% sulfuric acid (90 mL) at 5° C. was addeddropwise a solution of NaNO₂ (7.4 g, 107.25 mmol, 1.66 equiv) in water(15 mL). The reaction was stirred at 5° C. for another 30 min. Asolution of KI (32.1 g, 193.37 mmol, 3.00 equiv) in water (15 mL) wasadded dropwise at 5° C. The reaction was allowed to stir at 5° C. for 1h and then quenched by the addition of 50 mL of water. The precipitatewas collected by filtration and then dissolved in 150 mL of ethylacetate. The resulting solution was washed sequentially with 1×100 mL ofsaturated Na₂SO₃ solution, 1×100 mL of saturated sodium bicarbonatesolution and 1×100 mL of brine. The organic layer was dried overanhydrous sodium sulfate and concentrated under vacuum to give 10.8 g(63%) of ethyl 3-iodo-1H-pyrazole-4-carboxylate as a yellow solid. 1HNMR (300 MHz, CDCl₃): δ 8.18 (s, 1H), 4.38-4.29 (m, 2H), 1.41-1.33 (m,3H) ppm. LCMS (method B, ESI): RT=1.36 min, m/z=267.0 [M+H]+.

Step 2 ethyl3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate

A solution of ethyl 3-iodo-1H-pyrazole-4-carboxylate (10.8 g, 40.60mmol, 1.00 equiv), 3,4-dihydro-2H-pyran (10 g, 118.88 mmol, 2.93 equiv)and TsOH (780 mg, 4.53 mmol, 0.11 equiv) in THF (100 mL) was stirred for2 h at 60° C. The reaction mixture was cooled to room temperature andquenched by the addition of 100 mL of saturated sodium bicarbonatesolution. The resulting solution was extracted with 2×80 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with ethyl acetate/petroleum ether (1:20)to give 13 g (91%) of ethyl3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate as a yellow oil. 1H NMR(400 MHz, CDCl₃): δ 8.04 (s, 1H), 5.40-5.38 (m, 1H), 4.34-4.29 (m, 2H),4.08-4.05 (m, 1H), 3.73-3.70 (m, 1H), 2.07-1.98 (m, 3H), 1.69-1.62 (m,3H), 1.39-1.32 (m, 3H) ppm. LCMS (method C, ESI): RT=1.53 min, m/z=351.0[M+H]⁺.

Step 3 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylic acid

To a solution of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate(85 g, 242.75 mmol, 1.00 equiv) in THF (300 mL) and methanol (300 mL)was added a solution of LiOH (17.5 g, 730.69 mmol, 3.01 equiv) in water(400 mL). The resulting solution was stirred at room temperatureovernight and then concentrated under vacuum to remove the organicsolvent. The resulting solution was diluted with 400 mL of H₂O and thenacidified to pH 6.0 with 1M hydrochloric acid. The mixture was extractedwith 3×800 mL of dichloromethane. The combined organic layers was washedwith 3×1000 mL of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum to give 75 g (96%) of3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid as an off-whitesolid. LCMS (method D, ESI): RT=1.23 min, m/z=323.0 [M+H]⁺.

Step 4 (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol

To a solution of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid (28g, 86.93 mmol, 1.00 equiv) in anhydrous THF (300 mL) maintained undernitrogen at 5° C. was added a 1M solution of BH₃ in THF (300 mL)dropwise with stirring. The reaction was stirred overnight at roomtemperature and then quenched by the addition of 300 mL of saturatedNH₄Cl solution. The resulting mixture was extracted with 3×1000 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with ethyl acetate/petroleum ether (1:1)to give 12.67 g (47%) of (3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanolas a white solid. 1H NMR (400 MHz, DMSO-d6): δ 7.73 (s, 1H), 5.37-5.34(m, 1H), 4.92 (s, 1H), 4.20 (d, J=3.6 Hz, 2H), 3.89-3.88 (m, 1H),3.65-3.57 (m, 1H), 2.09-2.00 (m, 1H), 1.99-1.90 (m, 2H), 1.69-1.61 (m,1H), 1.49-1.46 (m, 2H) ppm. LCMS (method A, ESI): RT=1.16 min, m/z=309.0[M+H]⁺.

Step 5 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde

Into a 250-mL 3-necked round-bottom flask purged and. To a stirredsolution of oxalyl chloride (18.576 g, 146.35 mmol, 3.01 equiv) inanhydrous dichloromethane (300 mL) maintained under nitrogen at −78° C.was added DMSO (15.138 g, 193.75 mmol, 3.98 equiv) dropwise. Thereaction mixture was stirred at −65° C. for 30 min. A solution of(3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanol (15.0 g, 48.68 mmol, 1.00equiv) in dichloromethane (100 mL) was then added dropwise at −65° C.and the reaction was stirred for another 60 min at −65° C. Triethylamine(40.6 mL) was added dropwise at −65° C. and the reaction was stirred for30 min at −65° C. The reaction was warmed to 0° C. then quenched by theaddition of 100 mL of saturated NH₄Cl solution. The resulting mixturewas extracted with 3×400 mL of dichloromethane. The combined organiclayers was dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluted withethyl acetate/petroleum ether (1:20) to give 13.48 g (90%) of3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde as a golden oil. 1H NMR(300 MHz, DMSO-d6): δ 9.69 (s, 1H), 8.57 (s, 1H), 5.49 (dd, J=2.7 Hz,9.9 Hz, 1H), 3.95-3.91 (m, 1H), 3.68-3.62 (m, 1H), 2.11-2.01 (m, 3H),1.69-1.62 (m, 3H) ppm. LCMS (method A, ESI): RT=1.35 min, m/z=307.0[M+H]⁺.

Step 6 tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (21.5 g,70.24 mmol, 1.00 equiv), tert-butylN-methyl-N-(2-(methylamino)ethyl)carbamate (20 g, 106.23 mmol, 1.51equiv) and NaBH(OAc)₃ (29.8 g, 137.98 mmol, 1.96 equiv) indichloroethane (300 mL) was stirred for 1 h at room temperature. Thereaction was diluted with 300 mL of dichloromethane and then washed with3×300 mL of brine. The organic layer was dried over anhydrous sodiumsulfate and concentrated under vacuum. The residue was purified on asilica gel column eluted with 0-7% methanol in dichloromethane to give31 g (92%) of tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamateas a yellow oil. 1H NMR (300 MHz, CDCl₃): δ 7.62 (s, 1H), 5.34-5.30 (m,1H), 4.06-4.02 (m, 1H), 3.68-3.62 (m, 1H), 3.42-3.38 (m, 4H), 2.85 (s,4H), 2.62-2.53 (m, 2H), 2.47-2.46 (m, 2H), 2.13-1.97 (m, 3H), 1.74-1.69(m, 3H), 1.46 (s, 9H) ppm. LCMS (method A, ESI): RT=1.17 min, m/z=479.0[M+H]+.

Biological Methods PRMT1 Biochemical Assay

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates.

Peptide representative of human histone H4 residues 36-50 wassynthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide (SEQ ID NO.:1).

Molecular Biology:

Full-length human PRMT1 isoform 1 (NM_001536.5) transcript clone wasamplified from an HEK 293 cDNA library, incorporating flanking 5′sequence encoding a FLAG tag (DYKDDDDK) (SEQ ID NO.:2) fused directly toMet 1 of PRMT1. The amplified gene was subcloned into pFastBacI (LifeTechnologies) modified to encode an N-terminal GST tag and a TEVcleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYFQGGNS) (SEQ ID NO.:3)fused to Asp of the Flag tag of PRMT1.

Protein Expression.

Recombinant baculovirus were generated according to Bac-to-Bac kitinstructions (Life Technologies). Protein over-expression wasaccomplished by infecting exponentially growing High Five insect cellculture at 1.5×10⁶ cell/ml with 1:100 ratio of virus. Infections werecarried out at 27° C. for 48 hours, harvested by centrifugation, andstored at −80° C. for purification.

Protein Purification.

Expressed full-length human GST-tagged PRMT1 protein was purified fromcell paste by glutathione sepharose affinity chromatography afterequilibration of the resin with 50 mM phosphate buffer, 200 mM NaCl, 5%glycerol, 5 mM β-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT1 waseluted with 50 mM Tris, 2 mM glutathione, pH 7.8, dialysed in buffer Aand concentrated to 1 mg/mL. The purity of recovered protein was 73%.Reference: Wasilko, D. J. and S. E. Lee: “TIPS: titerless infected-cellspreservation and scale-up” Bioprocess J., 5 (2006), pp. 29-32.

Predicted Translations:

GST-tagged PRMT1 (SEQ ID NO.: 4)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYFQGGNSDYKDDDDKMAAAEAANCIMENFVATLANGMSLQPPLEEVSCGQAESSEKPNAEDMTSKDYYFDSYAHFGIHEEMLKDEVRTLTYRNSMFHNRHLFKDKVVLDVGSGTGILCMFAAKAGARKVIGIECSSISDYAVKIVKANKLDHVVTIIKGKVEEVELPVEKVDIIISEWMGYCLFYESMLNTVLYARDKWLAPDGLIFPDRATLYVTAIEDRQYKDYKIHWWENVYGFDMSCIKDVAIKEPLVDVVDPKQLVTNACLIKEVDIYTVKVEDLTFTSPFCLQVKRNDYVHALVAYFNIEFTRCHKRTGFSTSPESPYTHWKQTVFYMEDYLTVKTGEEIFGTIGMRPNAKNNRDLDFTIDLDFKGQLCEL SCSTDYRMR

General Procedure for PRMT1 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT1, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT1 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT1for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT1 was 0.5nM, ³H-SAM was 200 nM, non-radiolabeled SAM was 1.5 uM, peptide was 20nM, SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 300 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

%  inhibition  calculation${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{m\; i\; n}}{{dpm}_{{ma}\; x} - {dpm}_{m\; i\; n}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-parameter  IC₅₀  fit$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu} {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

PRMT6 Biochemical Assay

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine(SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin(BSG), sodium butyrate and Tris(2-carboxyethyl)phosphine hydrochloridesolution (TCEP) were purchased from Sigma-Aldrich at the highest levelof purity possible. ³H-SAM was purchase from American RadiolabeledChemicals with a specific activity of 80 Ci/mmol. 384-well streptavidinFlashplates were purchased from PerkinElmer.

Substrates.

Peptide representative of human histone H4 residues 36-50 wassynthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide and contained amonomethylated lysine at position 44 (SEQ ID NO.:5).

Molecular Biology:

Full-length human PRMT6 (NM_018137.2) transcript clone was amplifiedfrom an HEK 293 cDNA library, incorporating a flanking 5′ sequenceencoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:6) fused directly to Ser 2of PRMT6 and a 3′ sequence encoding a hexa His sequence (HHHHHH) fuseddirectly to Asp 375. The amplified gene was subcloned into pFastBacMam(Viva Biotech).

Protein Expression.

Recombinant baculovirus were generated according to Bac-to-Bac kitinstructions (Life Technologies). Protein over-expression wasaccomplished by infecting exponentially growing HEK 293F cell culture at1.3×10⁶ cell/ml with virus (MOI=10) in the presence of 8 mM sodiumbutyrate. Infections were carried out at 37° C. for 48 hours, harvestedby centrifugation, and stored at −80° C. for purification.

Protein Purification.

Expressed full-length human Flag- and His-tagged PRMT6 protein waspurified from cell paste by NiNTA agarose affinity chromatography afterequilibration of the resin with buffer containing 50 mM Tris, 300 mMNaCl, 10% glycerol, pH 7.8 (Buffer Ni-A). Column was washed with 20 mMimidazole in the same buffer and Flag-PRMT6-His was eluted with 150 mMimidazole. Pooled fractions were dialysed against buffer Ni-A andfurther purified by anti-flag M2 affinity chromatography. Flag-PRMT6-Hiswas eluted with 200 ug/ml FLAG peptide in the same buffer. Pooledfractions were dialysed in 20 mM Tris, 150 mM NaCl, 10% glycerol and 5mM β-mercaptoethanol, pH 7.8. The purity of recovered protein was 95%.

Predicted Translations:

Flag-PRMT6-His (SEQ ID NO.: 7)MDYKDDDDKSQPKKRKLESGGGGEGGEGTEEEDGAEREAALERPRRTKRERDQLYYECYSDVSVHEEMIADRVRTDAYRLGILRNWAALRGKTVLDVGAGTGILSIFCAQAGARRVYAVEASAIWQQAREVVRFNGLEDRVHVLPGPVETVELPEQVDAIVSEWMGYGLLHESMLSSVLHARTKWLKEGGLLLPASAELFIAPISDQMLEWRLGFWSQVKQHYGVDMSCLEGFATRCLMGHSEIVVQGLSGEDVLARPQRFAQLELSRAGLEQELEAGVGGRFRCSCYGSAPMHGFAIWFQVTFPGGESEKPLVLSTSPFHPATHWKQALLYLNEPVQVEQDTDVSGEITLLPSRDNPRRLRVLLRYKVGDQEEKTKDFAMEDHHHHHH

General Procedure for PRMT6 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT6, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT6 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT6for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT6 was 1 nM,³H-SAM was 200 nM, non-radiolabeled SAM was 250 nM, peptide was 75 nM,SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 400 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

%  inhibition  calculation${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{m\; i\; n}}{{dpm}_{{ma}\; x} - {dpm}_{m\; i\; n}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-parameter  IC₅₀  fit$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu} {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

PRMT8 Biochemical Assay

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG),isopropyl-β-D-thiogalactopyranoside (IPTG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates.

Peptide representative of human histone H4 residues 31-45 wassynthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-KPAIRRLARRGGVKR-amide (SEQ ID NO.:8).

Molecular Biology:

Full-length human PRMT8 (NM_019854.4) isoform 1 transcript clone wasamplified from an HEK 293 cDNA library and subcloned into pGEX-4T-1 (GELife Sciences). The resulting construct encodes an N-terminal GST tagand a thrombin cleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPEF) (SEQ ID NO.:9)fused directly to Met 1 of PRMT8.

Protein Expression.

E. coli (BL21(DE3) Gold, Stratagene) made competent by the CaCl₂ methodwere transformed with the PRMT8 construct and ampicillin selection.Protein over-expression was accomplished by growing the PRMT8 expressingE. coli clone and inducing expression with 0.3 mM IPTG at 16° C. Theculture was grown for 12 hours, harvested by centrifugation, and storedat −80° C. for purification.

Protein Purification.

Expressed full-length human GST-tagged PRMT8 protein was purified fromcell paste by glutathione sepharose affinity chromatography after theresin was equilibrated with 50 mM phosphate buffer, 200 mM NaCl, 5%glycerol, 5 mM β-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT8 waseluted with 50 mM Tris, 2 mM glutathione, pH 7.8. Pooled fractions werecleaved by thrombin (10 U) and dialysed in buffer A. GST was removed byreloading the cleaved protein sample onto glutathione sepharose columnand PRMT8 was collected in the flow-through fractions. PRMT8 waspurified further by ceramic hydroxyapatite chromatography. The columnwas washed with 50 mM phosphate buffer, 100 mM NaCl, 5% glycerol, 5 mMβ-mercaptoethanol, pH 7.8 and PRMT8 was eluted by 100 mM phosphate inthe same buffer. Protein was concentrated and buffer was exchanged to 50mM Tris, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, pH 7.8 byultrafiltration. The purity of recovered protein was 89%.

Predicted Translations:

GST-tagged PRMT8 (SEQ ID NO.: 10)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPEFMGMKHSSRCLLLRRKMAENAAESTEVNSPPSQPPQPVVPAKPVQCVHHVSTQPSCPGRGKMSKLLNPEEMTSRDYYFDSYAHFGIHEEMLKDEVRTLTYRNSMYHNKHVFKDKVVLDVGSGTGILSMFAAKAGAKKVFGIECSSISDYSEKIIKANHLDNIITIFKGKVEEVELPVEKVDIIISEWMGYCLFYESMLNTVIFARDKWLKPGGLMFPDRAALYVVAIEDRQYKDFKIHWWENVYGFDMTCIRDVAMKEPLVDIVDPKQVVTNACLIKEVDIYTVKTEELSFTSAFCLQIQRNDYVHALVTYFNIEFTKCHKKMGFSTAPDAPYTHWKQTVFYLEDYLTVRRGEEIYGTISMKPNAKNVRDLDFTVDLDFKGQLCETSVSNDYKMR

General Procedure for PRMT8 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT8, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT8 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT8for 30 min at room temperature, then a cocktail (10 ul) containing³H-SAM and peptide was added to initiate the reaction (final volume=51ul). The final concentrations of the components were as follows: PRMT8was 1.5 nM, ³H-SAM was 50 nM, non-radiolabeled SAM was 550 nM, peptidewas 150 nM, SAH in the minimum signal control wells was 1 mM, and theDMSO concentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 400 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

%  inhibition  calculation${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{m\; i\; n}}{{dpm}_{{ma}\; x} - {dpm}_{m\; i\; n}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-parameter  IC₅₀  fit$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu} {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

PRMT3 Biochemical Assay

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), ),isopropyl-β-D-thiogalactopyranoside (IPTG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates.

Peptide containing the classic RMT substrate motif was synthesized withan N-terminal linker-affinity tag motif and a C-terminal amide cap by21^(st) Century Biochemicals. The peptide was purified byhigh-performance liquid chromatography (HPLC) to greater than 95% purityand confirmed by liquid chromatography mass spectrometry (LC-MS). Thesequence was Biot-Ahx-GGRGGFGGRGGFGGRGGFG-amide (SEQ ID NO.:11).

Molecular Biology:

Full-length human PRMT3 (NM_005788.3) isoform 1 transcript clone wasamplified from an HEK 293 cDNA library and subcloned into pGEX-KG (GELife Sciences). The resulting construct encodes an N-terminal GST tagand a thrombin cleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS) (SEQ ID NO.:12)fused directly to Cys 2 of PRMT3.

Protein Expression.

E. coli (BL21(DE3) Gold, Stratagene) made competent by the CaCl₂ methodwere transformed with the PRMT3 construct and ampicillin selection.Protein over-expression was accomplished by growing the PRMT3 expressingE. coli clone and inducing expression with 0.3 mM IPTG at 16° C. Theculture was grown for 12 hours, harvested by centrifugation, and storedat −80° C. for purification.

Protein Purification.

Expressed full-length human GST-tagged PRMT3 protein was purified fromcell paste by glutathione sepharose affinity chromatography afterequilibration of the resin with 50 mM phosphate buffer, 200 mM NaCl, 5%glycerol, 1 mM EDTA, 5 mM β-mercaptoethanol, pH6.5 (Buffer A).GST-tagged PRMT3 was eluted with 50 mM Tris, 2 mM glutathione, pH 7.1and 50 mM Tris, 20 mM glutathione, pH 7.1. Pooled fractions weredialysed in 20 mM Tris, 50 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM DTT,pH7.5 (Buffer B) and applied to a Q Sepharose Fast Flow column.GST-tagged PRMT3 was eluted by 500 mM NaCl in buffer B. Pooled fractionswere dialyzed in 25 mM phosphate buffer, 100 mM NaCl, 5% glycerol, 2 mMDTT, pH 6.8 (Buffer C) and loaded on to a ceramic hydroxyapatite column.GST-tagged PRMT3 eluted with 25-400 mM phosphate in buffer C. Proteinwas concentrated and buffer was exchanged to 20 mM Tris, 150 mM NaCl, 5%glycerol, 5 mM β-mercaptoethanol, pH7.8 by ultrafiltration. The purityof recovered protein was 70%.

Predicted Translations:

GST-tagged PRMT3 (SEQ ID NO.: 13)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSCSLASGATGGRGAVENEEDLPELSDSGDEAAWEDEDDADLPHGKQQTPCLFCNRLFTSAEETFSHCKSEHQFNIDSMVHKHGLEFYGYIKLINFIRLKNPTVEYMNSIYNPVPWEKEEYLKPVLEDDLLLQFDVEDLYEPVSVPFSYPNGLSENTSVVEKLKHMEARALSAEAALARAREDLQKMKQFAQDFVMHTDVRTCSSSTSVIADLQEDEDGVYFSSYGHYGIHEEMLKDKIRTESYRDFIYQNPHIFKDKVVLDVGCGTGILSMFAAKAGAKKVLGVDQSEILYQAMDIIRLNKLEDTITLIKGKIEEVHLPVEKVDVIISEWMGYFLLFESMLDSVLYAKNKYLAKGGSVYPDICTISLVAVSDVNKHADRIAFWDDVYGFKMSCMKKAVIPEAVVEVLDPKTLISEPCGIKHIDCHTTSISDLEFSSDFTLKITRTSMCTAIAGYFDIYFEKNCHNRVVFSTGPQSTKTHWKQTVFLLEKPFSVKAGEALKGKVTVHKNKKDPRSLTVTLTLNNST QTYGLQ

General Procedure for PRMT3 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT3, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT3 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT3for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT3 was 0.5nM, ³H-SAM was 100 nM, non-radiolabeled SAM was 1.8 uM, peptide was 330nM, SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofpotassium chloride (10 ul) to a final concentration of 100 mM. 50 ul ofthe reaction in the 384-well polypropylene plate was then transferred toa 384-well Flashplate and the biotinylated peptides were allowed to bindto the streptavidin surface for at least 1 hour before being washed oncewith 0.1% Tween20 in a Biotek ELx405 plate washer. The plates were thenread in a PerkinElmer TopCount plate reader to measure the quantity of³H-labeled peptide bound to the Flashplate surface, measured asdisintegrations per minute (dpm) or alternatively, referred to as countsper minute (cpm).

%  inhibition  calculation${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{m\; i\; n}}{{dpm}_{{ma}\; x} - {dpm}_{m\; i\; n}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-parameter  IC₅₀  fit$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu} {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

CARM1 Biochemical Assay

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), sodiumbutyrate and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP)were purchased from Sigma-Aldrich at the highest level of puritypossible. ³H-SAM was purchase from American Radiolabeled Chemicals witha specific activity of 80 Ci/mmol. 384-well streptavidin Flashplateswere purchased from PerkinElmer.

Substrates.

Peptide representative of human histone H3 residues 16-30 wassynthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-PRKQLATKAARKSAP-amide and contained amonomethylated arginine at position 26 (SEQ ID NO.:14).

Molecular Biology:

Human CARM1 (PRMT4) (NM_199141.1) transcript clone was amplified from anHEK 293 cDNA library, incorporating a flanking 5′ sequence encoding aFLAG tag (MDYKDDDDK) (SEQ ID NO.:6) fused directly to Ala 2 of CARM1 and3′ sequence encoding a hexa His sequence (EGHHHHHH) (SEQ ID NO.:15)fused directly to Ser 608. The gene sequence encoding isoform1containing a deletion of amino acids 539-561 was amplified subsequentlyand subcloned into pFastBacMam (Viva Biotech).

Protein Expression.

Recombinant baculovirus were generated according to Bac-to-Bac kitinstructions (Life Technologies). Protein over-expression wasaccomplished by infecting exponentially growing HEK 293F cell culture at1.3×10⁶ cell/ml with virus (MOI=10) in the presence of 8 mM sodiumbutyrate. Infections were carried out at 37° C. for 48 hours, harvestedby centrifugation, and stored at −80° C. for purification.

Protein Purification.

Expressed full-length human Flag- and His-tagged CARM1 protein waspurified from cell paste by anti-flag M2 affinity chromatography withresin equilibrated with buffer containing 20 mM Tris, 150 mM NaCl, 5%glycerol, pH 7.8. Column was washed with 500 mM NaCl in buffer A andFlag-CARM1-His was eluted with 200 ug/ml FLAG peptide in buffer A.Pooled fractions were dialyzed in 20 mM Tris, 150 mM NaCl, 5% glyceroland 1 mM DTT, pH 7.8. The purity of recovered protein was 94.

Predicted Translations:

Flag-CARM1-His (SEQ ID NO.: 16)MDYKDDDDKAAAAAAVGPGAGGAGSAVPGGAGPCATVSVFPGARLLTIGDANGEIQRHAEQQALRLEVRAGPDSAGIALYSHEDVCVFKCSVSRETECSRVGKQSFIITLGCNSVLIQFATPNDFCSFYNILKTCRGHTLERSVFSERTEESSAVQYFQFYGYLSQQQNMMQDYVRTGTYQRAILQNHTDFKDKIVLDVGCGSGILSFFAAQAGARKIYAVEASTMAQHAEVLVKSNNLTDRIVVIPGKVEEVSLPEQVDIIISEPMGYMLFNERMLESYLHAKKYLKPSGNMFPTIGDVHLAPFTDEQLYMEQFTKANFWYQPSFHGVDLSALRGAAVDEYFRQPVVDTFDIRILMAKSVKYTVNFLEAKEGDLHRIEIPFKFHMLHSGLVHGLAFWFDVAFIGSIMTVWLSTAPTEPLTHWYQVRCLFQSPLFAKAGDTLSGTCLLIANKRQSYDISIVAQVDQTGSKSSNLLDLKNPFFRYTGTTPSPPPGSHYTSPSENMWNTGSTYNLSSGMAVAGMPTAYDLSSVIASGSSVGHNNLIPLGSSGAQGSGGGSTSAHYAVNSQFTMGGPAISMASPMSIPTNTMHYGSEGHHHHH H

General Procedure for CARM1 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of CARM1, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the CARM1 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with CARM1for 30 min at room temperature, then a cocktail (10 ul) containing³H-SAM and peptide was added to initiate the reaction (final volume=51ul). The final concentrations of the components were as follows: CARM1was 0.25 nM, ³H-SAM was 30 nM, peptide was 250 nM, SAH in the minimumsignal control wells was 1 mM, and the DMSO concentration was 2%. Theassays were stopped by the addition of non-radiolabeled SAM (10 ul) to afinal concentration of 300 uM, which dilutes the ³H-SAM to a level whereits incorporation into the peptide substrate is no longer detectable. 50ul of the reaction in the 384-well polypropylene plate was thentransferred to a 384-well Flashplate and the biotinylated peptides wereallowed to bind to the streptavidin surface for at least 1 hour beforebeing washed once with 0.1% Tween20 in a Biotek ELx405 plate washer. Theplates were then read in a PerkinElmer TopCount plate reader to measurethe quantity of ³H-labeled peptide bound to the Flashplate surface,measured as disintegrations per minute (dpm) or alternatively, referredto as counts per minute (cpm).

%  inhibition  calculation${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{m\; i\; n}}{{dpm}_{{ma}\; x} - {dpm}_{m\; i\; n}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-parameter  IC₅₀  fit$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu} {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

The biochemical evaluation of the exemplary compounds are shown in Table2.

TABLE 2 Biochemical evaluation Cmpd No. PRMT1 PRMT6 PRMT8 PRMT3 PRMT4 1A A B — — 2 A B B — — 3 A B — — — 4 A A — — — 5 A A — — — 6 A A — — — 7A A — — — 8 A A — — — 9 A A — — — 10 A B — — — 11 A A — — — 12 A A — — —13 A A — — — 14 A A — — — 17 A A — — — For Table 2, “A” indicates anIC₅₀ ≤0.100 μM, “B” indicates an IC₅₀ of 0.101-1.00 μM, “C” indicates anIC₅₀ of 1.01-3.00 μM, “D” indicates an IC₅₀ of 3.01-10 μM, and IC₅₀≥10.01 μM.

RKO Methylation Assay

RKO adherent cells were purchased from ATCC (American Type CultureCollection), Manassas, Va., USA. DMEM/Glutamax medium,penicillin-streptomycin, heat inactivated fetal bovine serum, 0.05%trypsin and D-PBS were purchased from Life Technologies, Grand Island,N.Y., USA. Odyssey blocking buffer, 800CW goat anti-rabbit IgG (H+L)antibody, and Licor Odyssey infrared scanner were purchased from LicorBiosciences, Lincoln, Nebr., USA. Mono-methyl arginine antibody waspurchased from Cell Signaling Technology, Danvers, Mass., USA. Methanolwas purchased from VWR, Franklin, Mass., USA. 10% Tween 20 was purchasedfrom KPL, Inc., Gaithersburg, Md., USA. DRAQ5 was purchased fromBiostatus Limited, Leicestershire, UK.

RKO adherent cells were maintained in growth medium (DMEM/Glutamaxmedium supplemented with 10% v/v heat inactivated fetal bovine serum and100 units/mL penicillin-streptomycin) and cultured at 37° C. under 5%CO₂.

Cell Treatment, in Cell Western (ICW) for Detection of Mono-MethylArginine and DNA Content.

RKO cells were seeded in assay medium at a concentration of 20,000 cellsper mL to a poly-D-lysine coated 384 well culture plate (BD Biosciences356697) with 50 μL per well. Compound (100 nL) from a 96-well sourceplate was added directly to 384 well cell plate. Plates were incubatedat 37° C., 5% CO₂ for 72 hours. After three days of incubation, plateswere brought to room temperature outside of the incubator for tenminutes and blotted on paper towels to remove cell media. 50 μL of icecold 100% methanol was added directly to each well and incubated for 30min at room temperature. After 30 min, plates were transferred to aBiotek EL406 plate washer and washed 2 times with 100 μL per well ofwash buffer (IX PBS). Next 60 μL per well of Odyssey blocking buffer(Odyssey Buffer with 0.1% Tween 20 (v/v)) were added to each plate andincubated 1 hour at room temperature. Blocking buffer was removed and 20μL per well of primary antibody was added (mono-methyl arginine diluted1:200 in Odyssey buffer with 0.1% Tween 20 (v/v)) and plates wereincubated overnight (16 hours) at 4° C. Plates were washed 5 times with100 μL per well of wash buffer. Next 20 μL per well of secondaryantibody was added (1:200 800CW goat anti-rabbit IgG (H+L) antibody,1:1000 DRAQ5 (Biostatus limited) in Odyssey buffer with 0.1% Tween 20(v/v)) and incubated for 1 hour at room temperature. The plates werewashed 5 times with 100 μL per well wash buffer then 2 times with 100 μLper well of water. Plates were allowed to dry at room temperature thenimaged on the Licor Odyssey machine which measures integrated intensityat 700 nm and 800 nm wavelengths. Both 700 and 800 channels werescanned.

Calculations:  First, the  ratio  for  each  well  was  determined  by:$\left( \frac{{monomethyl}\mspace{14mu} {Arginine}\mspace{14mu} 800\mspace{14mu} {nm}\mspace{14mu} {value}}{{DRAQ}\; 5\mspace{14mu} 700\mspace{14mu} {nm}\mspace{14mu} {value}} \right)$

Each plate included fourteen control wells of DMSO only treatment(minimum activation) as well as fourteen control wells for maximumactivation treated with 20 μM of a reference compound. The average ofthe ratio values for each control type was calculated and used todetermine the percent activation for each test well in the plate.Reference compound was serially diluted three-fold in DMSO for a totalof nine test concentrations, beginning at 20 μM. Percent activation wasdetermined and EC₃₀ curves were generated using triplicate wells perconcentration of compound.

${{Percent}\mspace{14mu} {Activation}} = {100 - \left( {\left( \frac{\begin{matrix}{\left( {{Individual}\mspace{14mu} {Test}\mspace{14mu} {Sample}\mspace{14mu} {Ratio}} \right) -} \\\left( {{Minimum}\mspace{14mu} {Activation}\mspace{14mu} {Ratio}} \right)\end{matrix}}{\begin{matrix}{\left( {{Maximum}\mspace{14mu} {Activation}\mspace{14mu} {Ratio}} \right) -} \\\left( {{Minimum}\mspace{14mu} {Activation}\mspace{14mu} {Ratio}} \right)\end{matrix}} \right)*100} \right)}$

TABLE 3 In Cell Western Data RGG ICW Cmpd No. EC30 1 A 2 B 3 C 4 A 5 B 6A 7 A 8 A 9 B 10 B 11 A 12 B 13 B 14 B 17 C For Table 3, “A” indicatesan EC₃₀ ≤3.00 μM, “B” indicates an EC₃₀ of 3.01-12.00 μM, and “C”indicates an EC₃₀ ≥12.01 μM.

OTHER EMBODIMENTS

The foregoing has been a description of certain non-limiting embodimentsof the invention. Those of ordinary skill in the art will appreciatethat various changes and modifications to this description may be madewithout departing from the spirit or scope of the present invention, asdefined in the following claims.

1.-30. (canceled)
 31. A kit or packaged pharmaceutical comprising acompound selected from:

and a pharmaceutically acceptable salt thereof, and instructions for usethereof.
 32. A method of inhibiting an arginine methyl tranferase (RMT)comprising contacting a cell with an effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof, wherein: X is NR₂ and Yis N; or X is N and Y is NR₂; each instance of R₁ is independentlyselected from the group consisting of hydrogen, halogen, —N₃, —CN, —NO₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂,—SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂; m is 0, 1,2, 3, or 4; R₂ is independently R₂ is hydrogen, optionally substitutedC₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substitutedC₂₋₆ alkynyl, optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl; or optionally substitutedC₁₋₄ alkyl-Cy; R₃ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄carbocylyl; R_(x) is independently optionally substituted C₁₋₄ alkyl oroptionally substituted C₃₋₄ carbocylyl; each instance of R^(A) isindependently selected from the group consisting of hydrogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, an oxygen protecting group when attached to an oxygen atom,and a sulfur protecting group when attached to a sulfur atom; eachinstance of R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted acyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, and a nitrogen protecting group, or twoR^(B) groups are taken together with their intervening atoms to form anoptionally substituted heterocyclic ring; and each instance of Cy isindependently optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl.
 33. The method of claim 32, whereinthe arginine methyl transferase is PRMT1, PRMT3, PRMT4, PRMT6, or PRMT8.34.-37. (canceled)
 38. A method of modulating gene expression comprisingcontacting a cell with an effective amount of a compound of Formula (I),or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof, wherein: X is NR₂ and Yis N; or X is N and Y is NR₂; each instance of R₁ is independentlyselected from the group consisting of hydrogen, halogen, —N₃, —CN, —NO₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂,—SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂; m is 0, 1,2, 3, or 4; R₂ is independently R₂ is hydrogen, optionally substitutedC₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substitutedC₂₋₆ alkynyl, optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl; or optionally substitutedC₁₋₄ alkyl-Cy; R₃ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄carbocylyl; R_(x) is independently optionally substituted C₁₋₄ alkyl oroptionally substituted C₃₋₄ carbocylyl; each instance of R^(A) isindependently selected from the group consisting of hydrogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, an oxygen protecting group when attached to an oxygen atom,and a sulfur protecting group when attached to a sulfur atom; eachinstance of R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted acyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, and a nitrogen protecting group, or twoR^(B) groups are taken together with their intervening atoms to form anoptionally substituted heterocyclic ring; and each instance of Cy isindependently optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl.
 39. A method of modulatingtranscription comprising contacting a cell with an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof, wherein: X is NR₂ and Yis N; or X is N and Y is NR₂; each instance of R₁ is independentlyselected from the group consisting of hydrogen, halogen, —N₃, —CN, —NO₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂,—SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R_(A), —NR_(B)SO₂R_(A), or —SO₂N(R^(B))₂; m is 0, 1,2, 3, or 4; R₂ is independently R₂ is hydrogen, optionally substitutedC₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substitutedC₂₋₆ alkynyl, optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl; or optionally substitutedC₁₋₄ alkyl-Cy; R₃ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄carbocylyl; R_(x) is independently optionally substituted C₁₋₄ alkyl oroptionally substituted C₃₋₄ carbocylyl; each instance of R^(A) isindependently selected from the group consisting of hydrogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, an oxygen protecting group when attached to an oxygen atom,and a sulfur protecting group when attached to a sulfur atom; eachinstance of R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted acyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, and a nitrogen protecting group, or twoR^(B) groups are taken together with their intervening atoms to form anoptionally substituted heterocyclic ring; and each instance of Cy isindependently optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl.
 40. (canceled)
 41. (canceled)
 42. Amethod of treating a RMT-mediated disorder, comprising administering toa subject in need thereof a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof, wherein: X is NR₂ and Yis N; or X is N and Y is NR₂; each instance of R₁ is independentlyselected from the group consisting of hydrogen, halogen, —N₃, —CN, —NO₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂,—SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R_(A), —SO₂R^(A), —NR_(B)SO₂R_(A), or —SO₂N(R^(B))₂; m is 0, 1,2, 3, or 4; R₂ is independently R₂ is hydrogen, optionally substitutedC₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substitutedC₂₋₆ alkynyl, optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl; or optionally substitutedC₁₋₄ alkyl-Cy; R₃ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄carbocylyl; R_(x) is independently optionally substituted C₁₋₄ alkyl oroptionally substituted C₃₋₄ carbocylyl; each instance of R^(A) isindependently selected from the group consisting of hydrogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, an oxygen protecting group when attached to an oxygen atom,and a sulfur protecting group when attached to a sulfur atom; eachinstance of R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted acyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, and a nitrogen protecting group, or twoR^(B) groups are taken together with their intervening atoms to form anoptionally substituted heterocyclic ring; and each instance of Cy isindependently optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl.
 43. The method of claim 42, whereinthe RMT-mediated disorder is a PRMT1-mediated disorder, a PRMT3-mediateddisorder, a PRMT4-mediated disorder, a PRMT6-mediated disorder, or aPRMT8-mediated disorder. 44.-47. (canceled)
 48. The method of claim 42,wherein the disorder is a proliferative disorder, a neurologicaldisorder, a muscular dystrophy, an autoimmune disorder, a vasculardisorder, or a metabolic disorder.
 49. The method of claim 42, whereinthe disorder is cancer.
 50. The method of claim 42, wherein the disorderis selected from the group consisting of breast cancer, prostate cancer,lung cancer, colon cancer, bladder cancer, lymphoma, leukemia, diabetesmellitus, kidney failure, coronary heart disease, oculopharyngealmuscular dystrophy, and amyotrophic lateral sclerosis.
 51. The method ofclaim 42, wherein the compound is of Formula (II-a):

or a pharmaceutically acceptable salt thereof, wherein: R₄ is optionallysubstituted C₁₋₆ alkyl, optionally substituted C₃₋₆ carbocyclyl,optionally substituted phenyl, optionally substituted 5- to 6-memberedheterocyclyl, optionally substituted 5- to 6-membered heteroaryl, oroptionally substituted C₁₋₆ alkyl-Cy.
 52. The method of claim 51,wherein R₄ is optionally substituted C₁₋₃ alkyl, optionally substitutedC₅ carbocyclyl, optionally substituted phenyl, optionally substituted 5-to 6-membered heteroaryl or optionally substituted C₁₋₃ alkyl-Cy,wherein Cy is optionally substituted phenyl or optionally substituted 5-to 6-membered heteroaryl.
 53. The method of claim 42, wherein thecompound is of Formula (II-a1):

or a pharmaceutically acceptable salt thereof, wherein: n is 1, 2, 3, 4,5, or 6; R₄ is selected from the group consisting of:

each instance of R₆ is independently halogen, —N₃, —CN, —NO₂, orC₁₋₄alkyl; and p is 0, 1, 2, or
 3. 54. The method of claim 42, whereinthe compound is of Formula (II-a2):

or a pharmaceutically acceptable salt thereof, wherein: each instance ofR₆ is independently halogen, —N₃, —CN, —NO₂, or C₁₋₄alkyl; R^(B) ishydrogen or C₁₋₄alkyl; n is 1, 2, 3, 4, 5, or 6; and q is 0, 1, or 2.55. The method of claim 42, wherein X is NR₂ and Y is N.
 56. The methodof claim 42, wherein X is N and Y is NR₂.
 57. The method of claim 42,wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 58. The method of claim42, wherein R_(x) is methyl, ethyl, isopropyl, isopropyl, propyl, butyl,hydroxyethyl, methoxyethyl, cyclopropyl, or cyclobutyl.
 59. The methodof claim 42, wherein R₃ is hydrogen, methyl, ethyl, propyl, butyl,cyclopropyl, or cyclobutyl.
 60. The method of claim 42, wherein thedisorder is breast cancer, prostate cancer, lung cancer, colon cancer,bladder cancer, or a leukemia.
 61. The method of claim 42, wherein thedisorder is a leukemia.
 62. The method of claim 61, wherein the leukemiais acute myelocytic leukemia.
 63. The method of claim 42, wherein thedisorder is a lymphoma.
 64. The method of claim 42, wherein the disorderis amyotrophic lateral sclerosis.
 65. The method of claim 42, whereinthe disorder is oculopharyngeal muscular dystrophy.
 66. The method ofclaim 42, wherein the disorder is diabetes.